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Biotechnology
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Biotechnology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in biotechnology.
ty, the labelled oligonucleotide remains bound to the M13 DNA, so subsequent binding to the beads results in retention of 33P in the well. Using this assay, over 200,000 compounds were screened and a novel helicase inhibitor was discovered.
Current Opinion in Biotechnology 1999, 10:105-111 http://biomednet.com/elecref/0958166901000105 © Elsevier Science Ltd ISSN 0958-1669 Contents (chosen by) 105 Analytical biotechnology (Tony Cass) 106 Plant biotechnology (Jim Dunwell) 106 Environmental biotechnology (Lawrence P Wackett) 107 Protein technologies and commercial enzymes (Gianfranco Gilardi) 109 Expression vectors and delivery systems (Thomas Kost and Patrick Condreay) 110 Pharmaceutical biotechnology (David L Pompliano)
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of special interest of outstanding interest
Analytical biotechnology Selected by Tony Cass Imperial College of Science, Technology and Medicine, London, UK
A piezoelectric biosensor as an olfactory receptor for odour detection: electronic nose. Wu TZ: Biosensors Bioelec 1999, 14:9-18. • Significance: Non-specific (or cross-reactive) sensing arrays are mimics of the animal olfactory system in that the analytical information is inherent in the pattern of responses of the array rather than the behaviour of any one element. These devices — ‘electronic noses’ — have used a range of sensor materials and, in combination with data processing methods, can analyse quite complex mixtures. Findings: An array of six piezoelectric (mass sensitive) elements was used in the device described in this paper. Five of the elements were coated with crude fractions of proteins isolated from frog olfactory cilia, and the sixth was a phospholipid-coated control. Exposure of the array to different volatile organic compounds gave rise to different shifts in the resonance frequency of the different elements resulting in a unique pattern of responses for each compound. High-throughput screening assay for helicase enzymes. Sivaraja M, Giordano H, Peterson MG: Anal Biochem 1998, 265:22-27. • Significance: Viral helicase enzymes are attractive targets for anti-infective agents as they play a vital part in DNA replication. High-throughput screening (HTS) of helicase activity would accelerate the discovery of novel inhibitors. This paper describes one assay approach suitable for this. Findings: An assay suitable for HTS in 96-well plates was established. It relied on the differential binding of high and low molecular weight DNA to glass beads. The helicase substrate comprised a 33P-labelled oligonucleotide annealed to M13 DNA. In the presence of active helicase, the labelled oligonucleotide is released through unwinding and the large M13 DNA binds to the glass beads, resulting in the loss of radioactivity from the well. In the presence of an inhibitor of helicase activi-
High-throughput quantitative histological analysis of Alzheimer’s disease pathology using a confocal digital microscanner. Hanzel DK, Trojanowski JQ, Johnston RF, Loring JF: Nat Biotechnol 1999, 17:53-57. •• Significance: High-throughput quantitative methods have not generally been applied to histological analysis of tissues but they would be of considerable utility in cases where rapid and objective results are required. Findings: Using a digital confocal microscanner, 2-colour digital images of brain tissue stained for amyloid protein were obtained in a few minutes with a pixel size of 10 mm x 10 mm and less than 1% cross talk between the two channels. In a mouse brain section, amyloid plaques could be identified in the images using automated feature extraction methods to eliminate background staining, and the extent of plaque formation could be quantified. This approach was also demonstrated to be applicable to human tissue. Engineering a regulatable enzyme for homogeneous immunoassays. Legendre D, Soumillion P, Fastrez J: Nat Biotechnol 1999, 17:67-71. •• Significance: Homogeneous immunoassays are particularly suitable for rapid assays as no blocking or washing steps are necessary. In one particular format, the catalytic activity of an enzyme–antigen conjugate is reduced by antibody binding. Such conjugates can, however, be tedious to prepare for each antigen and may not be consistent between preparations. This paper describes the construction of an enzyme–peptide engineered such that the enzyme is inhibited by antibody binding. Findings: Random peptide sequences (mimotopes) were inserted into surface loops of the enzyme TEM-1 β lactamase and the protein was subsequently displayed on the surface of phage. Selection of phage using monoclonal antibodies against prostate-specific antigen (PSA) yielded enzymes that bound the monoclonal antibody. These were subsequently screened for catalytic activity. The best enzymes were then used in a homogeneous competitive assay to detect PSA in the range 10–9 to 10–6 M. A microfabricated device for sizing and sorting DNA molecules. Chou H-P, Spence C, Scherer A, Quake S: Proc Natl Acad Sci USA 1999, 96:11-13. •• Significance: Conventional methods for sizing DNA molecules rely on their differential migration in an applied electric field. Although widely used, this has several disadvantages including its slow speed, the need for significant amounts of material and, because of the logarithmic dependence on molecular weight, low resolution with increasing size. Findings: DNA fragments ranging in size from 2 to 200 kbp were labelled with an intercalating fluorescent dye and flowed through a microfabricated T-structure, so that one molecule at a time was excited by laser irradiation. The fluorescence emission was imaged onto an avalanche photodiode and, as the number of dye molecules bound to the DNA is dependent on
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the latter’s length, the fluorescence intensity directly reflects the size of the molecule. Detection of individual oligonucleotide pairing by single molecule microscopy. Trabesinger W, Schulz GJ, Gruber HJ, Schindler H, Schmidt T: Anal Chem 1999, 71:279-283. •• Significance: DNA diagnostics, based on the hybridisation of a target sequence to a surface-bound probe sequence, has applications in mutation detection, genotyping and forensic science. Many different detection schemes have been proposed but most of them rely on fluorescence, usually by labelling the target DNA with a suitable dye and measuring the fluorescence intensity on the surface. The major limitation with intensity measurements arises from non-specific binding to the surface of the target. Using a dual label approach (one on the target, one on the streptavidin) excellent discrimination against non-specific binding can be achieved allowing for single molecule detection. Findings: Rhodamine-labelled streptavidin was bound at low density to a phospholipid-modified surface via a biotinylated lipid. The streptavidin subsequently captured a biotinylated target oligonucleotide which was subsequently detected with a cy5-labelled probe sequence. The fluorescence of the surface was measured at two wavelengths (corresponding to rhodamine and cy5) and coincident emission at the same point indicated a hybridisation event. This method is particularly effective in discriminating against non-specifically bound probe.
Plant biotechnology Selected by Jim Dunwell University of Reading, Berkshire, UK
Plants ectopically expressing the iron-binding protein, ferritin, are tolerant to oxidative damage and pathogens. Deak M, Horvath GV, Davletova S, Torok K, Sass L, Vass I, Barna B, Kiraly Z, Dudits D: Nat Biotechnol 1999, 17:192-196. •• Significance: This report suggests that sequestering intracellular iron involved in generation of reactive hydroxyl radicals may be a method of protecting plants from oxidative damage induced by a wide range of stresses. Findings: Transgenic tobacco which express an alfalfa ferritin gene either in plastids or in the cytoplasm were more tolerant to excess iron or paraquat treatment. Progeny from these plants were also more tolerant to necrotic damage caused by viral and fungal infection. They had normal photosynthetic function and chlorophyll content under glasshouse conditions. Overexpression of Pto activates defence responses and confers broad resistance. Tang X, Xie M, Kim YJ, Zhou J, Klessig DF, Martin GB: Plant Cell 1999, 11:15-29. • Significance: These findings provide evidence that by overexpressing specific disease-resistance genes it might be possible to engineer plants for durable resistance to a range of bacterial and fungal pathogens. Findings: The tomato disease (R) gene Pto provides race-specific resistance to the bacterial pathogen Pseudomonas syringae pv to tomato carrying the avrPto gene. Transgenic plants carrying the cauliflower mosaic virus 35S::Pto transgene showed activation of defence responses, and were more resistant to the pathogens P. syringae, Xanthomonas campestris, and Cladosporium fulvum than nontransgenic lines. Use of ubiquitin fusions to augment protein expression in transgenic plants. Hondred D, Walker JM, Mathews DE, Vierstra RD: Plant Physiol 1999, 119:713-724.
• Significance: This methods provides an improved system for the high-level expression of proteins in transgenic plants. Findings: The authors developed a set of vectors that express proteins and peptides as carboxy terminal translational fusions with ubiquitin. Studies with transgenic tobacco showed that such proteins are readily expressed to a high-level and that they are rapidly and precisely processed in vivo by ubiquitin specific proteases to release the fused protein moieties in free forms.
Environmental biotechnology Selected by Lawrence P Wackett University of Minnesota, St Paul, USA
Active efflux of organic solvents by Pseudomonas putida S12 is induced by solvents. Kieboom J, Dennis JJ, Zylstra GJ, deBont JAM: J Bacteriol 1998, 180:6769-6772. •• Significance: Bacteria survive high organic solvent fluxes in both natural and engineered environments. In the latter, bacterial fermentation processes that produce specialty chemicals may require growth in organic solvent–water two phase systems and they must survive such harsh conditions for optimal production of the target compound. A greater knowledge of how certain bacteria survive solvent stresses will allow use of such molecular systems in biotechnology. Findings: A region of DNA that acts as the promoter for the P. putida S12 solvent efflux pump was fused to a lacZ reporter system. Conditions that induce the efflux pump were then measured via β-galactosidase activity; using a convenient colorimetric assay. Metals and antibiotics did not induce the activity, many different solvents did induce the activities. Thus, the system is specific to hydrophobic liquids, and so it is specific to solvent stress. PHA synthase from Chromatium vinosum: cysteine 149 is involved in covalent catalysis. Muh U, Sinskey AJ, Kirby DP, Lane WS, Stubbe J: Biochemistry 1999, 38:826-837. • Significance: There is an enormous worldwide market for thermoplastics and increasing interest to make them with varying degrees of biodegradability. Polyhydroxyalkanoates (PHAs) can fill some of this market. Furthermore, they are derived from renewable resources, either bacteria (in which PHAs are produced naturally) or recombinant bacteria or plants. The enzyme that produces PHAs is a clear target for protein engineering, which will require better undertsanding of its reaction mechanism. Findings: Sequence alignments suggested the importance of several cysteine residues in the catalytic cycle of PHA synthase, the enzyme synthesizing PHAs. Site-directed mutagenesis and radiolabel trapping experiments suggested the role of at least one of the cysteine residues in covalent attachment of the growing PHA chain. Cold-active serine alkaline protease from the psychrotrophic bacterium Shewanella strain Ac10: gene cloning and enzyme purification and characterization. Kulakova L, Galkin A, Kurihara T, Yoshimura T, Esaki N: Appl Environ Microbiol 1999, 65:611-617. •• Significance: Proteases are prominent industrial enzymes. Their applications range from food processing to clothes washing. There has been a great deal of research on heat-stable proteases, but research on cold-active proteases will expand the range of biotechnological applications for this class of enzyme. Findings: The gene encoding a cold-active alkaline protease was cloned into E. coli and sequenced. The enzyme was shown to be related to the subtilisin family of proteases but it was five
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times more active in the temperature range 5–15oC than the industry standard subtilisin Carlsberg. Production of medium-chain-length poly(3-hydroxyalkanoates) from gluconate by recombinant Escherichia coli. Klinke S, Ren Q, Witholt B, Kessler B: Appl Environ Microbiol 1999, 65:540-548. •• Significance: There is increasing interest in using biodegradable polymers to prevent long-term accumulation of long-life-time polymers in the environment. A major class of biosynthesized, biodegradable polymers are the polyhydroxyalkanoates (PHAs). For a biotechnological process to be used, PHAs must be made from a cheap starting compound, such as gluconate. Findings: A recombinant E. coli containing PHA synthase that produces largely medium-chain-length PHAs was previously shown to use fatty acids as a starting material. In this report, a cytosolic thioesterase I gene was introduced into the E. coli. The strain was shown to produce up to 2.3% of cell dry weight of PHA with gluconate as the carbon source.
Protein technologies and commercial enzymes Selected by Gianfranco Gilardi Imperial College of Science, Technology and Medicine, London, UK
Evolutionary molecular engineering by random elongation mutagenesis. Matsuura T, Miyai K, Trakulnaleamsai S, Yomo T, Shima Y, Miki S, Yamamoto K, Urabe I: Nat Biotechnol 1999, 17:58-61. •• Significance: This paper reports a new method of random mutagenesis for molecular evolution strategies. Findings: The method involves the addition of peptides with random sequences, fused to the carboxy-terminal of the enzyme target. The method was successfully tested on a triple mutant of catalase I from Bacillus stearothermophilus that was shown to have lower thermostability compared to the wild-type enzyme. A 16 amino acid residue peptide, of which 10 amino acids were random, was fused to the carboxy-terminus of the catalase I triple mutant. The results showed a population of variants containing only mutants with higher thermostability than the triple mutant; some of them were more stable than the wild type. The results are interpreted in terms of an increase in the dimension of the sequence space that creates a new fitness landscape where further diversification of a property is allowed. Functionalised de novo designed proteins: mechanism for proton coupling to oxidation/reduction in heme protein maquettes. Shifman JM, Moser CC, Kalsbeck WA, Bocia DF, Dutton PL: Biochemistry 1998, 37:16815-16827. •• Significance: Proton pumping coupled to redox processes is a phenomenon essential to life, but not yet fully understood. This work reports on the successful synthesis of an artificial haem system able to mimic this phenomenon, offering a simple system where this phenomenon can be studied. Findings: A synthetic tetrahelix haem protein was synthesised to reproduce the natural proton coupling to haem redox activity. pK shifts between 4.2–7.0 and 9.4–10.3 were observed upon haem reduction, with an associated 210 mV decrease of the haem potential between the two extremes of pH. Glutamate residues were found to be the source of proton coupling below pH 8.0, whereas lysines were resposible for pH above 8.0. The protein core environment was found to be responsible for the pK shift of glutamate from 4.4 in solution to 7.0 in the protein. Furthermore, more than one glutamate residue needed to be removed before
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pH-independent redox activity could be observed, suggesting a distributed partial charge compensation. The magnitude of the partial charge compensation is influenced by the distance between the ferric haem and the amino acid sidechain. Rational design of a functional metalloenzyme: introduction of a site for manganese binding and oxidation into a heme peroxidase. Wilcox SK, Putnam CD, Sastry M, Blankenship J, Chazin WJ, McRee DE, Goodin DB: Biochemistry 1998, 37:16853-16862. •• Significance: This work is a novel approach to creating model metalloenzymes that use the scaffold of an existing redox-active haem enzyme. Findings: Functionally active models for manganese peroxidase (MnP) were created by creating a Mn-binding site in cytochrome c peroxidase (CcP). The X-ray structure of one of the models (MP6.8) showed that Mn2+ is coordinated to Asp45 and the haem 7-propionate, with a Kd of 0.2 mM. Reaction with hydrogen peroxide induces the oxidation of Mn2+ by electron trasfer to both the Trp191 radical and the ferryl haem centre. Molecular basis for interprotein complex-dependent effects on the redox properties of amicyanin. Zhu Z, Cunane LM, Chen Z, Durley RCE, Matthews FS, Davidson VL: Biochemistry 1998, 37:17128-17136. •• Significance: Methylamine dehydrogenase (MADH)–amicyanin–cytochrome c-i551I is one of best characterised physiological electron transfer complexes, also shown to be functional in the crystalline state. This work shows for the first time how protein–protein interactions in the complex affect the redox properties of amicyanin, with novel implications for rational design. Findings: MADH–amicyanin complex formation significantly lowers the oxidation-reduction midpoint potential (Em) of amicyanin. Surface charge neutralisation was ruled out by site-directed mutagenesis. Analysis of the X-ray structure of reduced amicyanin at pH 4.4 showed that His95, a ligand of the oxidised copper, is rotated by 180° around the Cß–Cγ bond relative to the oxidised structure, and it is no longer a copper ligand. The same reduced structure at pH 7.7 shows both the flipped structure observed at pH 4.4 and that observed for the oxidised protein at pH 4.8. When the flipped structure was modelled in that of the complex with MADH, the flipping was hindered by the complex, showing how the redox-linked, pHdependent rotation is controlled in the complex, hence affecting the redox properties. Effects of Asp-369 and Arg-372 mutations on heme environment and function in human endothelial nitric-oxide synthase. Chen P-F, Berka V, Tsai A-L, Wu KK: J Biol Chem 1998, 273:34164-34170. • Significance: Nitric oxide synthase is an important enzyme involved in many pathological and physiological processes. This work is a novel study on the structure-activity relationship of residues around the haem environment, in the putative substrate-binding region. Findings: Human endothelial nitric-oxide synthase (eNOS) was systematically mutated on residues Thr360, Arg365, Cys368, Asp369, Arg372, Tyr373, Glu377 and Asp378. Residues Asp369 and Arg372 were found to be essential for enzyme activity. All mutants, with exception of Asp369→Glu, Arg372→Lys and Arg372→Met, had low spin haem. Binding of L-arginine, (6Arg)-5,6,7,8-tetrahydro-L-biopetrin, 1-phenylimidazole, 3,5-lutidine and CO complexation was investigated in the different
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mutants; all results indicate that Asp369 and Arg372 play a critical role in the oxygenase domain structure and activity. Dynamics of protein-bound water in the heme domain of P450BM3 studied by high pressure spectroscopy: comparison with P450cam and P450 2B4. Davydov DR, Bon Hoa GH, Peterson JA: Biochemistry 1999, 38:751-761. •• Significance: Pressure-induced transitions in proteins combined with X-ray structural data is a powerful means to study the role of co-ordinated water molecules in redox proteins. This paper is a novel study on an important class of cytochromes with biotechnological relevance — cytochromes P450. Findings: Pressure-induced transitions in the haem domain of cytochrome P450 BM3 (P450BMP) were compared to those of Pseudomonas putida (P450cam) and rabbit liver microsomal 2B4 (2B4). Increase in hydrostatic pressure on BMP in the presence of palmitic acid caused a high-to-low spin transition and subsequent conversion of the P450 form into the inactive P420. The reaction volumes of the low-to-high spin transition of the cytochromes are consistent with the displacement of one water molecule. Larger volume changes were found when P450BMP and 2B4 bound their substrates (palmitic acid and benzphetamine) compared to P450cam (camphor), an their conversion from the high-to-low spin transition revealed a linear relationship with ∆G, suggesting a common mechanism for all three proteins. A synthetic peptide adhesion epitope as a novel antimicrobial agent. Kelly CG, Younson JS, Hikmat BY, Todryk SM, Czisch M, Haris PI, Flindall IR, Newby C, Mallet AI, Ma JK-C, Lehner T: Nat Biotechnol 1999, 17:42-47. • Significance: This paper reports a novel approach to rational design of competitive peptide inhibitors of adhesion, proposing a new strategy to prevent bacterial infection. Findings: A peptide (p1025) corresponding to residues 1025–1044 of adhesin was synthesised and its inhibitary activity towards binding of Streptococcus mutans to salivary receptors was studied in vitro by surface-plasmon resonance. Residues Glu1025 and Glu1037 were found to be important in binding by site-directed mutagenesis. Direct application of p1025 on teeth prevented colonisation of this microorganism. Oxygenation cascade in conversion of n-alkanes and α,ω ω-dioic acids catalysed by cytochrome P450 52A3. Scheller U, Zimmer T, Becher D, Schauer F, Schunck W-H: J Biol Chem 1998, 273:32528-32534. •• Significance: P450 enzymes offer a great potential in biotechnological applications. This paper shows how P450 monooxygenases may be involved not only in the primary, but also in subsequent oxidation steps that are of biotechnological interest. Findings: The activity of recombinant cytochrome P450 52A3 from Candida maltosa was studied using hexadecane as substrate. It was found that the primary product, 1-hexadecanol, was formed together with hexadecanal, hexadecanoic acid, 1,16-hexadecanediol, 16-hydroxydecanoic acid and 1,16-hexadecanedioic acid. The latter was also found to be a competitive inhibitor for νalkane binding, and may be involved in metabolic regulation. Different Vmax were found: 27, 23 and 69 min-1, for hexadecane, 1-hexadecanol and hexadecanal, respectively, and lower values, 9, 9 and 5 min-1, for hexadecanoic acid, 1,16-hexadecanediol and 16-hydroxyhexadecanoic acid, respectively. This work demonstrates that P450 can catalyse a cascade of sequential, monoand di-terminal monooxygentations with high regioselectivity.
Crystal structure of a plant catechol oxidase containing a dicopper center. Klabunde T, Eicken C, Sacchettini JC, Krebs B: Nat Struct Biol 1998, 5:1084-1090. •• Significance: This paper reports the structure of a novel, ubiquitous plant enzyme active on a wide range of o-diphenols, opening the possibility for rational design for variants of industrial interest. Findings: The resting dicupric Cu(II)-Cu(II), and the reduced dicuprous Cu(I)-Cu(I) structures of catechol oxidase were also studied in the presence of the inhibitor phenylthiourea. The dinuclear copper centre, type 3, was found in a central alpha-helical bundle, in a hydrophobic pocket near the surface. Copper ligands are three histidines for each copper; one histidine, His109, is covalently linked to Cys92. A mechanism is proposed where one of the oxygen atoms of the diphenolic substrate is bound to copper in the oxygenated enzyme. Improved immobilisation of fusion proteins via cellulosebinding domains. Linder M, Nevanen T, Soderholm L, Bengs O: Biotech Bioeng 1998, 60:642-647. •• Significance: The use of cellulose-binding domains (CBDs) in fusion proteins as tags for affinity purification and immobilisation is of great industrial interest. No pre-treatment is required, it is cheap, and chemically inert, hence safe for food and the pharmaceutical industry. This paper reports on a new strategy to increase immobilisation yields. Findings: CBD from variants of Trichoderma reesei cellobiohydrolase I were fused to antibody fragments. Conventional immobilisation resulted in a leakage of the immobilised protein from the column equivalent to 0.3–0.5% per column volume, as detected by tritium labelling. Leakage was significantly reduced (0.01% per column volume) by the fusion of two CBDs instead of a single one, bound via a linker. A sensitive photosystem II-based biosensor for detection of herbicides. Koblizek M, Masosjidek J, Komenda J, Kucera T, Pilloton R, Mattoo AK, Giardi MT: Biotech Bioeng 1998, 60:664-669. • Significance: Herbicides can be highly toxic to human and animal health; limits in drinking water are 0.1 mg/L of an individual pesticide or 0.5 mg/L of total pesticides. This paper reports on the successful use of photosystem II as a cheap and direct detection system of such compounds, opening new avenues for industrial applications. Findings: Photosystem II from the thermophilic cyanobacterium Synechococcus elongatus was immobilised on a Clark oxygen electrode mounted on a flow cell that was illuminated. Light-induced electron transfer causes the release of oxygen, which was inhibited in a concentration-dependent manner by the presence of herbicides. This system was successfully tested on diuron, atrazine, simazine, loxynil, bromoxynil, and dinoseb, showing detection limits in the range 10–8–10–10. Residual lignin studies of laccase-delignified kraft pulps. Sealey J, Ragauskas AJ: Enzyme Mirob Technol 1998, 23:422-426. • Significance: The industrial production of high-value paper products relies on the removal of lignin from wood. Chemical bleaching generates environmental concerns. This paper reports on a new strategy in the use of enzymatic processes for wood treatment. Findings: Laccase is well known to be able to be active on lignin, but its use in industrial applications has been limited by
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its inability to diffuse in the lignin network. This paper shows that the delignification properties of laccase are significantly improved by using N-hydroxybenzotriazole as a mediator able to shuttle electrons between the enzyme and the polymer. NMR and FTIR studies showed that this treatment oxidises the phenolic components of lignin that are found demethylated and enriched in carboxylic acid groups. Noncovalent immobilisation of chloroperoxidase onto talc: catalytic properties of a new biocatalyst. Aoun S, Chebli C, Baboulene M: Enzyme Microb Technol 1998, 23:380-385. • Significance: Enzymatic halogenation has not yet found industrial applications due to its high costs, stability and deactivation problems related to the enzymes used. This work reports on the successful immobilisation and activity improvement of one such enzyme on a cheap support. Findings: Chloroperoxidase from Caldariomyces fumago (CPO) is a haem-containing glycoprotein with haloperoxidase activity. Adsorption and activity studies were carried out on CPO immobilised on talc at pH 3.0 and pH 6.0. On this support, higher activity (60–70%) was found at pH 6.0. On the other hand, calcinated talc-type was found to be slightly less favourable to absorption, but showed excellent enzymatic activity, widening the potential industrial applications of this enzyme.
Expression vectors and dellivery systems Selected by Thomas Kost and Patrick Condreay GlaxoWellcome, North Carolina, USA
A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Piechaczek C, Fetzer C, Baiker A, Bode J, Lipps HJ: Nucleic Acids Res 1999, 27:426-428. • Significance: A vector is described that contains the SV40 origin of replication, yet is not dependent upon large T antigen to replicate independently in cells as an episomal element. This represents an important step towards a vector suitable for in vivo gene transfer applications, as it avoids the undesirable effects upon cell associated with SV40 large T antigen expression. Findings: Scaffold/matrix attached regions (S/MAR) are DNA sequences that are associated with chromosomal origins of replication. A vector was constructed containing an S/MAR and the SV40 origin of replication. When transfected into CHO cells, the vector can be isolated intact in Hirt extracts from G418-resistant cultures. There is no evidence of chromosomal integration, whereas previous studies have shown that vectors containing either cis-acting DNA element alone will integrate into the host cell genome. The episomal element persists at a low copy number in cells, even in the absence of selective pressure. Tetracycline-inducible expression systems with reduced basal activity in mammalian cells. Forster K, Helbi V, Lederer T, Urlinger S, Wittenburg N, Hillen W: Nucleic Acids Res 1999, 27:708-710. • Significance: Systems involving chimeric transactivating– DNA-binding proteins and artificial promoters have been used to achieve inducible gene expression in mammalian cells, though variable levels of basal transcription from these promoters are a limitation on their utility. This paper describes the introduction of chimeric transrepressors to decrease basal transcription and allow tighter regulation of gene expression. Findings: A mammalian repressor domain is fused to the DNAbinding domain of the tetracycline (tet) repressor to repress transcription from promoters containing tet operator sequences
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in the uninduced state. In one approach, the transactivator contains a DNA-binding domain that is reverse regulated by inducer (tet). In the absence of tet, the transrepressor is bound to the promoter, yet in the presence of the inducer the repressor releases and the transactivator binds the promoter. In another approach, the reverse-regulated transactivator has an altered DNA site specificity. The transrepressor binds at normal operator sites in the uninduced state, whereas in the induced state the activator binds to the mutant sites. Both systems reduce basal transcription approximately six fold while achieving several 100-fold activation when induced. Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Zanta MA, Belguise-Valladier P, Behr J-P: Proc Natl Acad Sci USA 1999, 96:91-96. •• Significance: Non-viral gene delivery systems require large amounts of DNA to effect gene expression due to poor translocation of DNA to the nucleus. This paper presents proof-of-principle that direct modification of DNA to exploit the cell’s nuclear translocation system will yield high levels of gene expression with reduced amounts of DNA. Findings: An expression cassette consisting of the CMV promoter and luciferase gene is covalently attached to a small peptide nuclear localization signal (NLS). The tagged construct elicits 10–1000-fold greater gene expression than the untagged construct in a variety of cell lines. A mutation in the NLS that abolishes nuclear transport does not exhibit the same enhancement when attached to DNA. Poxviral/retroviral chimeric vectors allow cytoplasmic production of transducing defective retroviral particles. Holzer GW, Mayrhofer JA, Gritschenberger W, Dorner F, Falkner FG: Virology 1999, 253:107-114. • Significance: This paper describes the use of vaccinia virus (VV) to generate transduction-competent retroviral particles. This is an important first step towards development of a VV vector to launch retroviral transduction for in vivo applications. Furthermore, this system can serve as a method for transient production of defective retroviruses. Findings: A retroviral genome was engineered to give optimal vaccinia-directed transcription in the cell cytoplasm and then placed into a recombinant VV. High titers of retroviral particles are transiently produced within eight hours of VV infection of a retrovirus packaging cell line, and these particles will stably transduce cell cultures. The VV-directed transient system produces significantly higher titers of retrovirus than transfection with plasmid, though the titers are not as high as those isolated from clonally selected stable producer cell lines. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Condreay JP, Witherspoon SM, Clay WC, Kost TA: Proc Natl Acad Sci USA 1999, 96:127-132. •• Significance: This report extends the reported mammalian cell host range of recombinant baculoviruses that utilize mammalian cell promoters and is the first demonstration that this system can be used for stable gene transfer in mammalian cells. Findings: A recombinant baculovirus was developed that contained the cytomegalovirus immediate early promoter/enhancer directing the expression of green fluorescent protein, and the SV40 virus early promoter controlling neomycin phosphotransferase II expression. Using this virus, efficient gene transfer and
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expression was observed in numerous cell types, including those of human origin. In all cases, the addition of butyrate or trichostatin A to the transduced cells markedly enhanced the level of reporter gene expression. The inclusion of the dominant selectable marker allowed for the selection of cell lines that stably maintain expression of the reporter gene for multiple passages. Adenovirus-mediated regulable target gene expression in vivo. Burcin MM, Schneider G, Kochanek S, Tsai SY, O’Malley BW: Proc Natl Acad Sci USA 1999, 96:355-360. •• Significance: The mifepristone-regulable recombinant adenovirus described in this paper provides an important tool for small-molecule regulated transgene expression in cultured cells and in vivo. Findings: A high capacity adenoviral vector devoid of all viral coding sequences was combined with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. The virus uses a chimeric transactivator, GLp65, consisting of a mutated progesterone receptor ligand-binding domain and part of the activation domain of the human p65 protein, a component of the NK-κB complex. Incorporation of the liver-specific transthyretin promoter into this vector system resulted in potent mifepristone-inducible expression of human growth hormone in both cultured HepG2 cells and in experimental mice. Defective vaccinia virus as a biologically safe tool for the overproduction of recombinant human secretory proteins. Himly M, Pfleiderer M, Holzer G, Fischer U, Hannak E, Falkner FG, Dorner F: Protein Expr Purif 1998, 14:317-326. • Significance: The recombinant vaccinia virus (VV) vector system, described here, provides improved safety and efficiency, while maintaining the ability to grow to high titers in a Complementing cell line. Findings: Recombinant VV based on a defective virus lacking the essential early transcribed gene coding for the enzyme uracil DNA glycosylase (VV D4R open reading frame) expressing cDNA’s coding for the human blood coagulation factors VII and IX produced significantly more recombinant protein than the corresponding recombinants based on wild-type VV. Infection of the complementing cell line RK-D4R-44.20 supported increased recombinant protein production compared to wild-type RK-13 cells. From a safety point of view, the reversion rate of the defective viruses was in the range of one revertant per 106 infectious particles., It was noted, however, that second generation defective viruses lacking any D4R sequences have been developed that abolish rare recombination events. Generation of destabilized green fluorescent protein as a transcription reporter. Li X, Zhao X, Fang Y, Jiang X, Fan C, Huang C-C, Kain SR: J Biol Chem 1998, 273:34970-34975. • Significance: The availability of a modified green flourescent protein (GFP) variant with a reduced half life extends the application of GFP as a reporter gene for those studies requiring rapid reporter turnover, such as transcriptional activation assays. Findings: Amino acids 422–461 of the degradation domain of mouse ornithine decarboxylase were fused to the carboxy-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike GFP, was unstable in the presence of cyclohexamide and had a half-life of two hours. Fluoresence decay of the fusion protein was found to correlate with protein degradation. The utility of EGFP as a transcription reporter was
demonstrated by linking it to NFκB binding sequences and monitoring tumor necrosis α-mediated NFκB activation. High-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli by fusion to the bacteriophage lambda head protein D. Forrer P, Jaussi R: Gene 1998, 224:45-52. • Significance: Histidine-tagged bacteriophage lambda head protein D (gpHD) is a small, cysteine-free monomeric protein that can serve as an efficient fusion partner for high level and soluble expression of heterologous proteins in E. coli cytoplasm. Findings: Various mammalian proteins, including Crk, JNK1, cJun, ATF-2, were fused to the carboxy-terminus of gpHD and expressed in E. coli. In all cases, the quantity of soluble fusion protein found in the cytoplasm was greater than that obtained when attempting to express the protein in the absence of the fusion partner. The gpHD c-Jun and ATF-2 fusion proteins were successfully used as substrates for mitogen-activated protein kinases. Various E.coli gene fusion expression systems exist. It will be interesting to follow the success rate obtained using the gpHD system with a variety of mammalian proteins. Two-helper RNA system for production of recombinant Semliki Forest virus particles. Smerdou C, Liljeström P: J Virol 1999, 73:1092-1098. • Significance: The development of a two-helper RNA system, described here, provides an efficient and higher biosafety profile system for the generation of recombinant Semliki Forest virus (SFV) particles. This may lead to more widespread use of this expression system. Findings: A new packaging system for SFV was developed. This system is based on the use of a two-helper system in which the capsid and spike proteins of the C-p62-6K-E1 viral polyprotein are expressed from two independent RNA molecules. Cotransfection of cells with both helper RNA’s and an SFV vector replicon carrying the LacZ gene led to the production of recombinant particles with titers up to 8 x 108 particles per 106 cells. No replication-proficient viruses could be detected in the recombinant virus preparations.
Pharmaceutical biotechnology Selected by David L Pompliano Merck Research Laboratories, Rahway, New Jersey
Reduced immunotoxicity and preservation of antibacterial activity in a releasable side-chain carbapenem antibiotic. Rosen H, Hajdu R, Silver L, Kropp H, Dorso K, Kohler J, Sundelof JG, Huber J, Hammond GG, Jackson JJ et al.: Science 1999, 283:703-706. •• Signficance: The intrinsic reactivity of β-lactams can lead to non-specific acylation of serum proteins. In turn, these haptenated proteins can stimulate an immune response resulting in unwanted side effects. This paper presents an elegant solution to this problem. By mechanistically coupling the expulsion of an antigenic sidechain to the opening of the β-lactam ring, the immunotoxic effects can be functionally separated from the antibacterial effects. Findings: Comparison of the immunoreactivity of several structural classes of carbapenems led to the conclusion that both humoral and cellular immunotoxic responses were due to an immunodominant 2-position sidechain epitope and not to the carbapenem nucleus itself. The sidechain is required for optimal binding to the bacterial target proteins. Carbapenem L-786,392 was designed so that β-lactam ring opening was linked chemically to release of
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the 2-position sidechain. This molecule was extremely effective against clinically problematic bacterial strains (MRSA and VanR Enterococci) in both in vitro and in animal models of infection. HPLC analysis confirmed the release of the sidechain upon enzyme-catalyzed ring opening. No pathological changes (anemia or lymphocytosis) in rhesus monkeys were observed after four weeks of L-786,392 at doses up to 100 mg/kg/day. The sidechain moiety showed no effect in hemagluttination assays (which are a measure of immunoreactivity) at high doses. Host control of HIV-1 parasitism in T cells by the nuclear factor of activated T cells. Kinoshita S, Chen BK, Kaneshima H, Nolan GP: Cell 1998, 95:595-604. • Significance: The action of nuclear factor of transcription (NFATc), a host regulator protein essential for the activation of T cells, is also required for productive HIV-1 infection in primary CD4+ T cells. Inhibiting the cellular processes activated by NFATc could lead to novel anti-HIV therapies for which resistance would be difficult to acquire. Findings: It was known that HIV-1 can replicate in PHA-stimulated primary T cells but not in quiescent T cells. The authors determined that PHA-stimulation of primary peripheral blood mononuclear cells (PBMCs) leads to increased expression of NFATc but not of NFkB (another cellular factor associated with T cell activation). When CD4+ T cells expressing NFATc (from retroviral gene transfer) were infected with HIV-1, complete reverse transcription of the viral genome ensued in the absence of PHA stimulation, showing that expression of NFATc activates the required cellular proceses to replicate the viral genome. Treatment of cells expressing NFATc with cyclosporin (CsA) or FK-506, drugs that inhibit the activation of NFATc, blocks complete HIV-1 reverse transcription. Thus, HIV-1 depends upon existing host processes that are activated by NFATc. Interleukin-13: central mediator of allergic asthma. WillsKarp M, Luyimbazi J, Xu X, Schofield B, Neben TY, Karp CL, Donaldson DD: Science 1998, 282:2258-2261. AND
Requirement for IL-13 independently of IL-4 in experimental asthma. Brunig G, Warnock M, Wakil AE, Venkayya R, Brombacher FB, Rennick DM, Sheppard D, Mohrs M, Donaldson DD, Locksley RM, Corry DB: Science 1998, 282:2261-2263.
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• Significance: The incidence and severity of allergic asthma are increasing. The discovery that IL-13 is a crucial component of the etiology of this disease will focus new drug therapies on blocking the action of this cytokine. Findings: In mouse models of allergic asthma, blocking IL-13 action was achieved by administering a soluble version of the IL-13 receptor. This fusion protein, IL-13α2–IgGFc, is known to neutralize IL-13, but not IL-4 (another cytokine implicated in allergic asthma), by binding to IL-13 and preventing it from interacting with its cell-surface receptor. Allergen-induced airway tightening and excess mucus production, referred to as airway hyperresponsiveness (AHR), is inhibited in mice treated with the soluble IL-13 receptor. IL-13 administered to mice in the absence of allergen challenge still induced AHR, showing a causal role for IL-13 in this asthma model. Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro. Fisher JE, Rogers MJ, Halasy JM, Luckman SP, Hughes DE, Masarachia PJ, Wesolowski G, Russell RGG, Rodan GA, Reszka AA: Proc Natl Acad Sci USA 1999, 96:133-138. • Significance: Alendronate (ALN), a bisphosphonate (BP), is used clinically to prevent osteoporosis. While ALN and other BPs inhibit osteoclast activity, their precise molecular target(s) are unknown. Thus, the unraveling of their mechanism of action, begun in this paper, will aid in the selection of new drug targets and better guide the discovery of treatments for osteoporosis. Findings: Earlier experiments had shown that ALN inhibits cholesterol biosynthesis, and that exogeneous addition of mevalonate, farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) can reverse the blockade. To sort out which step of the mevalonate pathway is the key target of ALN, a more complete set of pathway intermediates were tested for their ability to reverse the effects of ALN in osteoclasts. Geranylgeraniol (GGOH) proved to be the most effective antagonist of ALN inhibition. ALN-treated osteoclasts show a seven fold increase in the activity of a 34 kDa kinase compared to untreated controls. This activation is blocked by GGOH. Note that farnesol and squalene are without effect. Thus, the authors propose that ALN inhibits a GGPP-forming step, which probably affects the prenylation by GGPP of key regulatory proteins in the cell.
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Biotechnology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in biotechnology.
ty, the labelled oligonucleotide remains bound to the M13 DNA, so subsequent binding to the beads results in retention of 33P in the well. Using this assay, over 200,000 compounds were screened and a novel helicase inhibitor was discovered.
Current Opinion in Biotechnology 1999, 10:105-111 http://biomednet.com/elecref/0958166901000105 © Elsevier Science Ltd ISSN 0958-1669 Contents (chosen by) 105 Analytical biotechnology (Tony Cass) 106 Plant biotechnology (Jim Dunwell) 106 Environmental biotechnology (Lawrence P Wackett) 107 Protein technologies and commercial enzymes (Gianfranco Gilardi) 109 Expression vectors and delivery systems (Thomas Kost and Patrick Condreay) 110 Pharmaceutical biotechnology (David L Pompliano)
• ••
of special interest of outstanding interest
Analytical biotechnology Selected by Tony Cass Imperial College of Science, Technology and Medicine, London, UK
A piezoelectric biosensor as an olfactory receptor for odour detection: electronic nose. Wu TZ: Biosensors Bioelec 1999, 14:9-18. • Significance: Non-specific (or cross-reactive) sensing arrays are mimics of the animal olfactory system in that the analytical information is inherent in the pattern of responses of the array rather than the behaviour of any one element. These devices — ‘electronic noses’ — have used a range of sensor materials and, in combination with data processing methods, can analyse quite complex mixtures. Findings: An array of six piezoelectric (mass sensitive) elements was used in the device described in this paper. Five of the elements were coated with crude fractions of proteins isolated from frog olfactory cilia, and the sixth was a phospholipid-coated control. Exposure of the array to different volatile organic compounds gave rise to different shifts in the resonance frequency of the different elements resulting in a unique pattern of responses for each compound. High-throughput screening assay for helicase enzymes. Sivaraja M, Giordano H, Peterson MG: Anal Biochem 1998, 265:22-27. • Significance: Viral helicase enzymes are attractive targets for anti-infective agents as they play a vital part in DNA replication. High-throughput screening (HTS) of helicase activity would accelerate the discovery of novel inhibitors. This paper describes one assay approach suitable for this. Findings: An assay suitable for HTS in 96-well plates was established. It relied on the differential binding of high and low molecular weight DNA to glass beads. The helicase substrate comprised a 33P-labelled oligonucleotide annealed to M13 DNA. In the presence of active helicase, the labelled oligonucleotide is released through unwinding and the large M13 DNA binds to the glass beads, resulting in the loss of radioactivity from the well. In the presence of an inhibitor of helicase activi-
High-throughput quantitative histological analysis of Alzheimer’s disease pathology using a confocal digital microscanner. Hanzel DK, Trojanowski JQ, Johnston RF, Loring JF: Nat Biotechnol 1999, 17:53-57. •• Significance: High-throughput quantitative methods have not generally been applied to histological analysis of tissues but they would be of considerable utility in cases where rapid and objective results are required. Findings: Using a digital confocal microscanner, 2-colour digital images of brain tissue stained for amyloid protein were obtained in a few minutes with a pixel size of 10 mm x 10 mm and less than 1% cross talk between the two channels. In a mouse brain section, amyloid plaques could be identified in the images using automated feature extraction methods to eliminate background staining, and the extent of plaque formation could be quantified. This approach was also demonstrated to be applicable to human tissue. Engineering a regulatable enzyme for homogeneous immunoassays. Legendre D, Soumillion P, Fastrez J: Nat Biotechnol 1999, 17:67-71. •• Significance: Homogeneous immunoassays are particularly suitable for rapid assays as no blocking or washing steps are necessary. In one particular format, the catalytic activity of an enzyme–antigen conjugate is reduced by antibody binding. Such conjugates can, however, be tedious to prepare for each antigen and may not be consistent between preparations. This paper describes the construction of an enzyme–peptide engineered such that the enzyme is inhibited by antibody binding. Findings: Random peptide sequences (mimotopes) were inserted into surface loops of the enzyme TEM-1 β lactamase and the protein was subsequently displayed on the surface of phage. Selection of phage using monoclonal antibodies against prostate-specific antigen (PSA) yielded enzymes that bound the monoclonal antibody. These were subsequently screened for catalytic activity. The best enzymes were then used in a homogeneous competitive assay to detect PSA in the range 10–9 to 10–6 M. A microfabricated device for sizing and sorting DNA molecules. Chou H-P, Spence C, Scherer A, Quake S: Proc Natl Acad Sci USA 1999, 96:11-13. •• Significance: Conventional methods for sizing DNA molecules rely on their differential migration in an applied electric field. Although widely used, this has several disadvantages including its slow speed, the need for significant amounts of material and, because of the logarithmic dependence on molecular weight, low resolution with increasing size. Findings: DNA fragments ranging in size from 2 to 200 kbp were labelled with an intercalating fluorescent dye and flowed through a microfabricated T-structure, so that one molecule at a time was excited by laser irradiation. The fluorescence emission was imaged onto an avalanche photodiode and, as the number of dye molecules bound to the DNA is dependent on
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the latter’s length, the fluorescence intensity directly reflects the size of the molecule. Detection of individual oligonucleotide pairing by single molecule microscopy. Trabesinger W, Schulz GJ, Gruber HJ, Schindler H, Schmidt T: Anal Chem 1999, 71:279-283. •• Significance: DNA diagnostics, based on the hybridisation of a target sequence to a surface-bound probe sequence, has applications in mutation detection, genotyping and forensic science. Many different detection schemes have been proposed but most of them rely on fluorescence, usually by labelling the target DNA with a suitable dye and measuring the fluorescence intensity on the surface. The major limitation with intensity measurements arises from non-specific binding to the surface of the target. Using a dual label approach (one on the target, one on the streptavidin) excellent discrimination against non-specific binding can be achieved allowing for single molecule detection. Findings: Rhodamine-labelled streptavidin was bound at low density to a phospholipid-modified surface via a biotinylated lipid. The streptavidin subsequently captured a biotinylated target oligonucleotide which was subsequently detected with a cy5-labelled probe sequence. The fluorescence of the surface was measured at two wavelengths (corresponding to rhodamine and cy5) and coincident emission at the same point indicated a hybridisation event. This method is particularly effective in discriminating against non-specifically bound probe.
Plant biotechnology Selected by Jim Dunwell University of Reading, Berkshire, UK
Plants ectopically expressing the iron-binding protein, ferritin, are tolerant to oxidative damage and pathogens. Deak M, Horvath GV, Davletova S, Torok K, Sass L, Vass I, Barna B, Kiraly Z, Dudits D: Nat Biotechnol 1999, 17:192-196. •• Significance: This report suggests that sequestering intracellular iron involved in generation of reactive hydroxyl radicals may be a method of protecting plants from oxidative damage induced by a wide range of stresses. Findings: Transgenic tobacco which express an alfalfa ferritin gene either in plastids or in the cytoplasm were more tolerant to excess iron or paraquat treatment. Progeny from these plants were also more tolerant to necrotic damage caused by viral and fungal infection. They had normal photosynthetic function and chlorophyll content under glasshouse conditions. Overexpression of Pto activates defence responses and confers broad resistance. Tang X, Xie M, Kim YJ, Zhou J, Klessig DF, Martin GB: Plant Cell 1999, 11:15-29. • Significance: These findings provide evidence that by overexpressing specific disease-resistance genes it might be possible to engineer plants for durable resistance to a range of bacterial and fungal pathogens. Findings: The tomato disease (R) gene Pto provides race-specific resistance to the bacterial pathogen Pseudomonas syringae pv to tomato carrying the avrPto gene. Transgenic plants carrying the cauliflower mosaic virus 35S::Pto transgene showed activation of defence responses, and were more resistant to the pathogens P. syringae, Xanthomonas campestris, and Cladosporium fulvum than nontransgenic lines. Use of ubiquitin fusions to augment protein expression in transgenic plants. Hondred D, Walker JM, Mathews DE, Vierstra RD: Plant Physiol 1999, 119:713-724.
• Significance: This methods provides an improved system for the high-level expression of proteins in transgenic plants. Findings: The authors developed a set of vectors that express proteins and peptides as carboxy terminal translational fusions with ubiquitin. Studies with transgenic tobacco showed that such proteins are readily expressed to a high-level and that they are rapidly and precisely processed in vivo by ubiquitin specific proteases to release the fused protein moieties in free forms.
Environmental biotechnology Selected by Lawrence P Wackett University of Minnesota, St Paul, USA
Active efflux of organic solvents by Pseudomonas putida S12 is induced by solvents. Kieboom J, Dennis JJ, Zylstra GJ, deBont JAM: J Bacteriol 1998, 180:6769-6772. •• Significance: Bacteria survive high organic solvent fluxes in both natural and engineered environments. In the latter, bacterial fermentation processes that produce specialty chemicals may require growth in organic solvent–water two phase systems and they must survive such harsh conditions for optimal production of the target compound. A greater knowledge of how certain bacteria survive solvent stresses will allow use of such molecular systems in biotechnology. Findings: A region of DNA that acts as the promoter for the P. putida S12 solvent efflux pump was fused to a lacZ reporter system. Conditions that induce the efflux pump were then measured via β-galactosidase activity; using a convenient colorimetric assay. Metals and antibiotics did not induce the activity, many different solvents did induce the activities. Thus, the system is specific to hydrophobic liquids, and so it is specific to solvent stress. PHA synthase from Chromatium vinosum: cysteine 149 is involved in covalent catalysis. Muh U, Sinskey AJ, Kirby DP, Lane WS, Stubbe J: Biochemistry 1999, 38:826-837. • Significance: There is an enormous worldwide market for thermoplastics and increasing interest to make them with varying degrees of biodegradability. Polyhydroxyalkanoates (PHAs) can fill some of this market. Furthermore, they are derived from renewable resources, either bacteria (in which PHAs are produced naturally) or recombinant bacteria or plants. The enzyme that produces PHAs is a clear target for protein engineering, which will require better undertsanding of its reaction mechanism. Findings: Sequence alignments suggested the importance of several cysteine residues in the catalytic cycle of PHA synthase, the enzyme synthesizing PHAs. Site-directed mutagenesis and radiolabel trapping experiments suggested the role of at least one of the cysteine residues in covalent attachment of the growing PHA chain. Cold-active serine alkaline protease from the psychrotrophic bacterium Shewanella strain Ac10: gene cloning and enzyme purification and characterization. Kulakova L, Galkin A, Kurihara T, Yoshimura T, Esaki N: Appl Environ Microbiol 1999, 65:611-617. •• Significance: Proteases are prominent industrial enzymes. Their applications range from food processing to clothes washing. There has been a great deal of research on heat-stable proteases, but research on cold-active proteases will expand the range of biotechnological applications for this class of enzyme. Findings: The gene encoding a cold-active alkaline protease was cloned into E. coli and sequenced. The enzyme was shown to be related to the subtilisin family of proteases but it was five
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times more active in the temperature range 5–15oC than the industry standard subtilisin Carlsberg. Production of medium-chain-length poly(3-hydroxyalkanoates) from gluconate by recombinant Escherichia coli. Klinke S, Ren Q, Witholt B, Kessler B: Appl Environ Microbiol 1999, 65:540-548. •• Significance: There is increasing interest in using biodegradable polymers to prevent long-term accumulation of long-life-time polymers in the environment. A major class of biosynthesized, biodegradable polymers are the polyhydroxyalkanoates (PHAs). For a biotechnological process to be used, PHAs must be made from a cheap starting compound, such as gluconate. Findings: A recombinant E. coli containing PHA synthase that produces largely medium-chain-length PHAs was previously shown to use fatty acids as a starting material. In this report, a cytosolic thioesterase I gene was introduced into the E. coli. The strain was shown to produce up to 2.3% of cell dry weight of PHA with gluconate as the carbon source.
Protein technologies and commercial enzymes Selected by Gianfranco Gilardi Imperial College of Science, Technology and Medicine, London, UK
Evolutionary molecular engineering by random elongation mutagenesis. Matsuura T, Miyai K, Trakulnaleamsai S, Yomo T, Shima Y, Miki S, Yamamoto K, Urabe I: Nat Biotechnol 1999, 17:58-61. •• Significance: This paper reports a new method of random mutagenesis for molecular evolution strategies. Findings: The method involves the addition of peptides with random sequences, fused to the carboxy-terminal of the enzyme target. The method was successfully tested on a triple mutant of catalase I from Bacillus stearothermophilus that was shown to have lower thermostability compared to the wild-type enzyme. A 16 amino acid residue peptide, of which 10 amino acids were random, was fused to the carboxy-terminus of the catalase I triple mutant. The results showed a population of variants containing only mutants with higher thermostability than the triple mutant; some of them were more stable than the wild type. The results are interpreted in terms of an increase in the dimension of the sequence space that creates a new fitness landscape where further diversification of a property is allowed. Functionalised de novo designed proteins: mechanism for proton coupling to oxidation/reduction in heme protein maquettes. Shifman JM, Moser CC, Kalsbeck WA, Bocia DF, Dutton PL: Biochemistry 1998, 37:16815-16827. •• Significance: Proton pumping coupled to redox processes is a phenomenon essential to life, but not yet fully understood. This work reports on the successful synthesis of an artificial haem system able to mimic this phenomenon, offering a simple system where this phenomenon can be studied. Findings: A synthetic tetrahelix haem protein was synthesised to reproduce the natural proton coupling to haem redox activity. pK shifts between 4.2–7.0 and 9.4–10.3 were observed upon haem reduction, with an associated 210 mV decrease of the haem potential between the two extremes of pH. Glutamate residues were found to be the source of proton coupling below pH 8.0, whereas lysines were resposible for pH above 8.0. The protein core environment was found to be responsible for the pK shift of glutamate from 4.4 in solution to 7.0 in the protein. Furthermore, more than one glutamate residue needed to be removed before
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pH-independent redox activity could be observed, suggesting a distributed partial charge compensation. The magnitude of the partial charge compensation is influenced by the distance between the ferric haem and the amino acid sidechain. Rational design of a functional metalloenzyme: introduction of a site for manganese binding and oxidation into a heme peroxidase. Wilcox SK, Putnam CD, Sastry M, Blankenship J, Chazin WJ, McRee DE, Goodin DB: Biochemistry 1998, 37:16853-16862. •• Significance: This work is a novel approach to creating model metalloenzymes that use the scaffold of an existing redox-active haem enzyme. Findings: Functionally active models for manganese peroxidase (MnP) were created by creating a Mn-binding site in cytochrome c peroxidase (CcP). The X-ray structure of one of the models (MP6.8) showed that Mn2+ is coordinated to Asp45 and the haem 7-propionate, with a Kd of 0.2 mM. Reaction with hydrogen peroxide induces the oxidation of Mn2+ by electron trasfer to both the Trp191 radical and the ferryl haem centre. Molecular basis for interprotein complex-dependent effects on the redox properties of amicyanin. Zhu Z, Cunane LM, Chen Z, Durley RCE, Matthews FS, Davidson VL: Biochemistry 1998, 37:17128-17136. •• Significance: Methylamine dehydrogenase (MADH)–amicyanin–cytochrome c-i551I is one of best characterised physiological electron transfer complexes, also shown to be functional in the crystalline state. This work shows for the first time how protein–protein interactions in the complex affect the redox properties of amicyanin, with novel implications for rational design. Findings: MADH–amicyanin complex formation significantly lowers the oxidation-reduction midpoint potential (Em) of amicyanin. Surface charge neutralisation was ruled out by site-directed mutagenesis. Analysis of the X-ray structure of reduced amicyanin at pH 4.4 showed that His95, a ligand of the oxidised copper, is rotated by 180° around the Cß–Cγ bond relative to the oxidised structure, and it is no longer a copper ligand. The same reduced structure at pH 7.7 shows both the flipped structure observed at pH 4.4 and that observed for the oxidised protein at pH 4.8. When the flipped structure was modelled in that of the complex with MADH, the flipping was hindered by the complex, showing how the redox-linked, pHdependent rotation is controlled in the complex, hence affecting the redox properties. Effects of Asp-369 and Arg-372 mutations on heme environment and function in human endothelial nitric-oxide synthase. Chen P-F, Berka V, Tsai A-L, Wu KK: J Biol Chem 1998, 273:34164-34170. • Significance: Nitric oxide synthase is an important enzyme involved in many pathological and physiological processes. This work is a novel study on the structure-activity relationship of residues around the haem environment, in the putative substrate-binding region. Findings: Human endothelial nitric-oxide synthase (eNOS) was systematically mutated on residues Thr360, Arg365, Cys368, Asp369, Arg372, Tyr373, Glu377 and Asp378. Residues Asp369 and Arg372 were found to be essential for enzyme activity. All mutants, with exception of Asp369→Glu, Arg372→Lys and Arg372→Met, had low spin haem. Binding of L-arginine, (6Arg)-5,6,7,8-tetrahydro-L-biopetrin, 1-phenylimidazole, 3,5-lutidine and CO complexation was investigated in the different
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mutants; all results indicate that Asp369 and Arg372 play a critical role in the oxygenase domain structure and activity. Dynamics of protein-bound water in the heme domain of P450BM3 studied by high pressure spectroscopy: comparison with P450cam and P450 2B4. Davydov DR, Bon Hoa GH, Peterson JA: Biochemistry 1999, 38:751-761. •• Significance: Pressure-induced transitions in proteins combined with X-ray structural data is a powerful means to study the role of co-ordinated water molecules in redox proteins. This paper is a novel study on an important class of cytochromes with biotechnological relevance — cytochromes P450. Findings: Pressure-induced transitions in the haem domain of cytochrome P450 BM3 (P450BMP) were compared to those of Pseudomonas putida (P450cam) and rabbit liver microsomal 2B4 (2B4). Increase in hydrostatic pressure on BMP in the presence of palmitic acid caused a high-to-low spin transition and subsequent conversion of the P450 form into the inactive P420. The reaction volumes of the low-to-high spin transition of the cytochromes are consistent with the displacement of one water molecule. Larger volume changes were found when P450BMP and 2B4 bound their substrates (palmitic acid and benzphetamine) compared to P450cam (camphor), an their conversion from the high-to-low spin transition revealed a linear relationship with ∆G, suggesting a common mechanism for all three proteins. A synthetic peptide adhesion epitope as a novel antimicrobial agent. Kelly CG, Younson JS, Hikmat BY, Todryk SM, Czisch M, Haris PI, Flindall IR, Newby C, Mallet AI, Ma JK-C, Lehner T: Nat Biotechnol 1999, 17:42-47. • Significance: This paper reports a novel approach to rational design of competitive peptide inhibitors of adhesion, proposing a new strategy to prevent bacterial infection. Findings: A peptide (p1025) corresponding to residues 1025–1044 of adhesin was synthesised and its inhibitary activity towards binding of Streptococcus mutans to salivary receptors was studied in vitro by surface-plasmon resonance. Residues Glu1025 and Glu1037 were found to be important in binding by site-directed mutagenesis. Direct application of p1025 on teeth prevented colonisation of this microorganism. Oxygenation cascade in conversion of n-alkanes and α,ω ω-dioic acids catalysed by cytochrome P450 52A3. Scheller U, Zimmer T, Becher D, Schauer F, Schunck W-H: J Biol Chem 1998, 273:32528-32534. •• Significance: P450 enzymes offer a great potential in biotechnological applications. This paper shows how P450 monooxygenases may be involved not only in the primary, but also in subsequent oxidation steps that are of biotechnological interest. Findings: The activity of recombinant cytochrome P450 52A3 from Candida maltosa was studied using hexadecane as substrate. It was found that the primary product, 1-hexadecanol, was formed together with hexadecanal, hexadecanoic acid, 1,16-hexadecanediol, 16-hydroxydecanoic acid and 1,16-hexadecanedioic acid. The latter was also found to be a competitive inhibitor for νalkane binding, and may be involved in metabolic regulation. Different Vmax were found: 27, 23 and 69 min-1, for hexadecane, 1-hexadecanol and hexadecanal, respectively, and lower values, 9, 9 and 5 min-1, for hexadecanoic acid, 1,16-hexadecanediol and 16-hydroxyhexadecanoic acid, respectively. This work demonstrates that P450 can catalyse a cascade of sequential, monoand di-terminal monooxygentations with high regioselectivity.
Crystal structure of a plant catechol oxidase containing a dicopper center. Klabunde T, Eicken C, Sacchettini JC, Krebs B: Nat Struct Biol 1998, 5:1084-1090. •• Significance: This paper reports the structure of a novel, ubiquitous plant enzyme active on a wide range of o-diphenols, opening the possibility for rational design for variants of industrial interest. Findings: The resting dicupric Cu(II)-Cu(II), and the reduced dicuprous Cu(I)-Cu(I) structures of catechol oxidase were also studied in the presence of the inhibitor phenylthiourea. The dinuclear copper centre, type 3, was found in a central alpha-helical bundle, in a hydrophobic pocket near the surface. Copper ligands are three histidines for each copper; one histidine, His109, is covalently linked to Cys92. A mechanism is proposed where one of the oxygen atoms of the diphenolic substrate is bound to copper in the oxygenated enzyme. Improved immobilisation of fusion proteins via cellulosebinding domains. Linder M, Nevanen T, Soderholm L, Bengs O: Biotech Bioeng 1998, 60:642-647. •• Significance: The use of cellulose-binding domains (CBDs) in fusion proteins as tags for affinity purification and immobilisation is of great industrial interest. No pre-treatment is required, it is cheap, and chemically inert, hence safe for food and the pharmaceutical industry. This paper reports on a new strategy to increase immobilisation yields. Findings: CBD from variants of Trichoderma reesei cellobiohydrolase I were fused to antibody fragments. Conventional immobilisation resulted in a leakage of the immobilised protein from the column equivalent to 0.3–0.5% per column volume, as detected by tritium labelling. Leakage was significantly reduced (0.01% per column volume) by the fusion of two CBDs instead of a single one, bound via a linker. A sensitive photosystem II-based biosensor for detection of herbicides. Koblizek M, Masosjidek J, Komenda J, Kucera T, Pilloton R, Mattoo AK, Giardi MT: Biotech Bioeng 1998, 60:664-669. • Significance: Herbicides can be highly toxic to human and animal health; limits in drinking water are 0.1 mg/L of an individual pesticide or 0.5 mg/L of total pesticides. This paper reports on the successful use of photosystem II as a cheap and direct detection system of such compounds, opening new avenues for industrial applications. Findings: Photosystem II from the thermophilic cyanobacterium Synechococcus elongatus was immobilised on a Clark oxygen electrode mounted on a flow cell that was illuminated. Light-induced electron transfer causes the release of oxygen, which was inhibited in a concentration-dependent manner by the presence of herbicides. This system was successfully tested on diuron, atrazine, simazine, loxynil, bromoxynil, and dinoseb, showing detection limits in the range 10–8–10–10. Residual lignin studies of laccase-delignified kraft pulps. Sealey J, Ragauskas AJ: Enzyme Mirob Technol 1998, 23:422-426. • Significance: The industrial production of high-value paper products relies on the removal of lignin from wood. Chemical bleaching generates environmental concerns. This paper reports on a new strategy in the use of enzymatic processes for wood treatment. Findings: Laccase is well known to be able to be active on lignin, but its use in industrial applications has been limited by
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its inability to diffuse in the lignin network. This paper shows that the delignification properties of laccase are significantly improved by using N-hydroxybenzotriazole as a mediator able to shuttle electrons between the enzyme and the polymer. NMR and FTIR studies showed that this treatment oxidises the phenolic components of lignin that are found demethylated and enriched in carboxylic acid groups. Noncovalent immobilisation of chloroperoxidase onto talc: catalytic properties of a new biocatalyst. Aoun S, Chebli C, Baboulene M: Enzyme Microb Technol 1998, 23:380-385. • Significance: Enzymatic halogenation has not yet found industrial applications due to its high costs, stability and deactivation problems related to the enzymes used. This work reports on the successful immobilisation and activity improvement of one such enzyme on a cheap support. Findings: Chloroperoxidase from Caldariomyces fumago (CPO) is a haem-containing glycoprotein with haloperoxidase activity. Adsorption and activity studies were carried out on CPO immobilised on talc at pH 3.0 and pH 6.0. On this support, higher activity (60–70%) was found at pH 6.0. On the other hand, calcinated talc-type was found to be slightly less favourable to absorption, but showed excellent enzymatic activity, widening the potential industrial applications of this enzyme.
Expression vectors and dellivery systems Selected by Thomas Kost and Patrick Condreay GlaxoWellcome, North Carolina, USA
A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Piechaczek C, Fetzer C, Baiker A, Bode J, Lipps HJ: Nucleic Acids Res 1999, 27:426-428. • Significance: A vector is described that contains the SV40 origin of replication, yet is not dependent upon large T antigen to replicate independently in cells as an episomal element. This represents an important step towards a vector suitable for in vivo gene transfer applications, as it avoids the undesirable effects upon cell associated with SV40 large T antigen expression. Findings: Scaffold/matrix attached regions (S/MAR) are DNA sequences that are associated with chromosomal origins of replication. A vector was constructed containing an S/MAR and the SV40 origin of replication. When transfected into CHO cells, the vector can be isolated intact in Hirt extracts from G418-resistant cultures. There is no evidence of chromosomal integration, whereas previous studies have shown that vectors containing either cis-acting DNA element alone will integrate into the host cell genome. The episomal element persists at a low copy number in cells, even in the absence of selective pressure. Tetracycline-inducible expression systems with reduced basal activity in mammalian cells. Forster K, Helbi V, Lederer T, Urlinger S, Wittenburg N, Hillen W: Nucleic Acids Res 1999, 27:708-710. • Significance: Systems involving chimeric transactivating– DNA-binding proteins and artificial promoters have been used to achieve inducible gene expression in mammalian cells, though variable levels of basal transcription from these promoters are a limitation on their utility. This paper describes the introduction of chimeric transrepressors to decrease basal transcription and allow tighter regulation of gene expression. Findings: A mammalian repressor domain is fused to the DNAbinding domain of the tetracycline (tet) repressor to repress transcription from promoters containing tet operator sequences
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in the uninduced state. In one approach, the transactivator contains a DNA-binding domain that is reverse regulated by inducer (tet). In the absence of tet, the transrepressor is bound to the promoter, yet in the presence of the inducer the repressor releases and the transactivator binds the promoter. In another approach, the reverse-regulated transactivator has an altered DNA site specificity. The transrepressor binds at normal operator sites in the uninduced state, whereas in the induced state the activator binds to the mutant sites. Both systems reduce basal transcription approximately six fold while achieving several 100-fold activation when induced. Gene delivery: a single nuclear localization signal peptide is sufficient to carry DNA to the cell nucleus. Zanta MA, Belguise-Valladier P, Behr J-P: Proc Natl Acad Sci USA 1999, 96:91-96. •• Significance: Non-viral gene delivery systems require large amounts of DNA to effect gene expression due to poor translocation of DNA to the nucleus. This paper presents proof-of-principle that direct modification of DNA to exploit the cell’s nuclear translocation system will yield high levels of gene expression with reduced amounts of DNA. Findings: An expression cassette consisting of the CMV promoter and luciferase gene is covalently attached to a small peptide nuclear localization signal (NLS). The tagged construct elicits 10–1000-fold greater gene expression than the untagged construct in a variety of cell lines. A mutation in the NLS that abolishes nuclear transport does not exhibit the same enhancement when attached to DNA. Poxviral/retroviral chimeric vectors allow cytoplasmic production of transducing defective retroviral particles. Holzer GW, Mayrhofer JA, Gritschenberger W, Dorner F, Falkner FG: Virology 1999, 253:107-114. • Significance: This paper describes the use of vaccinia virus (VV) to generate transduction-competent retroviral particles. This is an important first step towards development of a VV vector to launch retroviral transduction for in vivo applications. Furthermore, this system can serve as a method for transient production of defective retroviruses. Findings: A retroviral genome was engineered to give optimal vaccinia-directed transcription in the cell cytoplasm and then placed into a recombinant VV. High titers of retroviral particles are transiently produced within eight hours of VV infection of a retrovirus packaging cell line, and these particles will stably transduce cell cultures. The VV-directed transient system produces significantly higher titers of retrovirus than transfection with plasmid, though the titers are not as high as those isolated from clonally selected stable producer cell lines. Transient and stable gene expression in mammalian cells transduced with a recombinant baculovirus vector. Condreay JP, Witherspoon SM, Clay WC, Kost TA: Proc Natl Acad Sci USA 1999, 96:127-132. •• Significance: This report extends the reported mammalian cell host range of recombinant baculoviruses that utilize mammalian cell promoters and is the first demonstration that this system can be used for stable gene transfer in mammalian cells. Findings: A recombinant baculovirus was developed that contained the cytomegalovirus immediate early promoter/enhancer directing the expression of green fluorescent protein, and the SV40 virus early promoter controlling neomycin phosphotransferase II expression. Using this virus, efficient gene transfer and
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expression was observed in numerous cell types, including those of human origin. In all cases, the addition of butyrate or trichostatin A to the transduced cells markedly enhanced the level of reporter gene expression. The inclusion of the dominant selectable marker allowed for the selection of cell lines that stably maintain expression of the reporter gene for multiple passages. Adenovirus-mediated regulable target gene expression in vivo. Burcin MM, Schneider G, Kochanek S, Tsai SY, O’Malley BW: Proc Natl Acad Sci USA 1999, 96:355-360. •• Significance: The mifepristone-regulable recombinant adenovirus described in this paper provides an important tool for small-molecule regulated transgene expression in cultured cells and in vivo. Findings: A high capacity adenoviral vector devoid of all viral coding sequences was combined with a regulatory system that can be used to express a target gene in vivo in a selected site and at a desired time. The virus uses a chimeric transactivator, GLp65, consisting of a mutated progesterone receptor ligand-binding domain and part of the activation domain of the human p65 protein, a component of the NK-κB complex. Incorporation of the liver-specific transthyretin promoter into this vector system resulted in potent mifepristone-inducible expression of human growth hormone in both cultured HepG2 cells and in experimental mice. Defective vaccinia virus as a biologically safe tool for the overproduction of recombinant human secretory proteins. Himly M, Pfleiderer M, Holzer G, Fischer U, Hannak E, Falkner FG, Dorner F: Protein Expr Purif 1998, 14:317-326. • Significance: The recombinant vaccinia virus (VV) vector system, described here, provides improved safety and efficiency, while maintaining the ability to grow to high titers in a Complementing cell line. Findings: Recombinant VV based on a defective virus lacking the essential early transcribed gene coding for the enzyme uracil DNA glycosylase (VV D4R open reading frame) expressing cDNA’s coding for the human blood coagulation factors VII and IX produced significantly more recombinant protein than the corresponding recombinants based on wild-type VV. Infection of the complementing cell line RK-D4R-44.20 supported increased recombinant protein production compared to wild-type RK-13 cells. From a safety point of view, the reversion rate of the defective viruses was in the range of one revertant per 106 infectious particles., It was noted, however, that second generation defective viruses lacking any D4R sequences have been developed that abolish rare recombination events. Generation of destabilized green fluorescent protein as a transcription reporter. Li X, Zhao X, Fang Y, Jiang X, Fan C, Huang C-C, Kain SR: J Biol Chem 1998, 273:34970-34975. • Significance: The availability of a modified green flourescent protein (GFP) variant with a reduced half life extends the application of GFP as a reporter gene for those studies requiring rapid reporter turnover, such as transcriptional activation assays. Findings: Amino acids 422–461 of the degradation domain of mouse ornithine decarboxylase were fused to the carboxy-terminal end of an enhanced variant of GFP (EGFP). The fusion protein, unlike GFP, was unstable in the presence of cyclohexamide and had a half-life of two hours. Fluoresence decay of the fusion protein was found to correlate with protein degradation. The utility of EGFP as a transcription reporter was
demonstrated by linking it to NFκB binding sequences and monitoring tumor necrosis α-mediated NFκB activation. High-level expression of soluble heterologous proteins in the cytoplasm of Escherichia coli by fusion to the bacteriophage lambda head protein D. Forrer P, Jaussi R: Gene 1998, 224:45-52. • Significance: Histidine-tagged bacteriophage lambda head protein D (gpHD) is a small, cysteine-free monomeric protein that can serve as an efficient fusion partner for high level and soluble expression of heterologous proteins in E. coli cytoplasm. Findings: Various mammalian proteins, including Crk, JNK1, cJun, ATF-2, were fused to the carboxy-terminus of gpHD and expressed in E. coli. In all cases, the quantity of soluble fusion protein found in the cytoplasm was greater than that obtained when attempting to express the protein in the absence of the fusion partner. The gpHD c-Jun and ATF-2 fusion proteins were successfully used as substrates for mitogen-activated protein kinases. Various E.coli gene fusion expression systems exist. It will be interesting to follow the success rate obtained using the gpHD system with a variety of mammalian proteins. Two-helper RNA system for production of recombinant Semliki Forest virus particles. Smerdou C, Liljeström P: J Virol 1999, 73:1092-1098. • Significance: The development of a two-helper RNA system, described here, provides an efficient and higher biosafety profile system for the generation of recombinant Semliki Forest virus (SFV) particles. This may lead to more widespread use of this expression system. Findings: A new packaging system for SFV was developed. This system is based on the use of a two-helper system in which the capsid and spike proteins of the C-p62-6K-E1 viral polyprotein are expressed from two independent RNA molecules. Cotransfection of cells with both helper RNA’s and an SFV vector replicon carrying the LacZ gene led to the production of recombinant particles with titers up to 8 x 108 particles per 106 cells. No replication-proficient viruses could be detected in the recombinant virus preparations.
Pharmaceutical biotechnology Selected by David L Pompliano Merck Research Laboratories, Rahway, New Jersey
Reduced immunotoxicity and preservation of antibacterial activity in a releasable side-chain carbapenem antibiotic. Rosen H, Hajdu R, Silver L, Kropp H, Dorso K, Kohler J, Sundelof JG, Huber J, Hammond GG, Jackson JJ et al.: Science 1999, 283:703-706. •• Signficance: The intrinsic reactivity of β-lactams can lead to non-specific acylation of serum proteins. In turn, these haptenated proteins can stimulate an immune response resulting in unwanted side effects. This paper presents an elegant solution to this problem. By mechanistically coupling the expulsion of an antigenic sidechain to the opening of the β-lactam ring, the immunotoxic effects can be functionally separated from the antibacterial effects. Findings: Comparison of the immunoreactivity of several structural classes of carbapenems led to the conclusion that both humoral and cellular immunotoxic responses were due to an immunodominant 2-position sidechain epitope and not to the carbapenem nucleus itself. The sidechain is required for optimal binding to the bacterial target proteins. Carbapenem L-786,392 was designed so that β-lactam ring opening was linked chemically to release of
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the 2-position sidechain. This molecule was extremely effective against clinically problematic bacterial strains (MRSA and VanR Enterococci) in both in vitro and in animal models of infection. HPLC analysis confirmed the release of the sidechain upon enzyme-catalyzed ring opening. No pathological changes (anemia or lymphocytosis) in rhesus monkeys were observed after four weeks of L-786,392 at doses up to 100 mg/kg/day. The sidechain moiety showed no effect in hemagluttination assays (which are a measure of immunoreactivity) at high doses. Host control of HIV-1 parasitism in T cells by the nuclear factor of activated T cells. Kinoshita S, Chen BK, Kaneshima H, Nolan GP: Cell 1998, 95:595-604. • Significance: The action of nuclear factor of transcription (NFATc), a host regulator protein essential for the activation of T cells, is also required for productive HIV-1 infection in primary CD4+ T cells. Inhibiting the cellular processes activated by NFATc could lead to novel anti-HIV therapies for which resistance would be difficult to acquire. Findings: It was known that HIV-1 can replicate in PHA-stimulated primary T cells but not in quiescent T cells. The authors determined that PHA-stimulation of primary peripheral blood mononuclear cells (PBMCs) leads to increased expression of NFATc but not of NFkB (another cellular factor associated with T cell activation). When CD4+ T cells expressing NFATc (from retroviral gene transfer) were infected with HIV-1, complete reverse transcription of the viral genome ensued in the absence of PHA stimulation, showing that expression of NFATc activates the required cellular proceses to replicate the viral genome. Treatment of cells expressing NFATc with cyclosporin (CsA) or FK-506, drugs that inhibit the activation of NFATc, blocks complete HIV-1 reverse transcription. Thus, HIV-1 depends upon existing host processes that are activated by NFATc. Interleukin-13: central mediator of allergic asthma. WillsKarp M, Luyimbazi J, Xu X, Schofield B, Neben TY, Karp CL, Donaldson DD: Science 1998, 282:2258-2261. AND
Requirement for IL-13 independently of IL-4 in experimental asthma. Brunig G, Warnock M, Wakil AE, Venkayya R, Brombacher FB, Rennick DM, Sheppard D, Mohrs M, Donaldson DD, Locksley RM, Corry DB: Science 1998, 282:2261-2263.
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• Significance: The incidence and severity of allergic asthma are increasing. The discovery that IL-13 is a crucial component of the etiology of this disease will focus new drug therapies on blocking the action of this cytokine. Findings: In mouse models of allergic asthma, blocking IL-13 action was achieved by administering a soluble version of the IL-13 receptor. This fusion protein, IL-13α2–IgGFc, is known to neutralize IL-13, but not IL-4 (another cytokine implicated in allergic asthma), by binding to IL-13 and preventing it from interacting with its cell-surface receptor. Allergen-induced airway tightening and excess mucus production, referred to as airway hyperresponsiveness (AHR), is inhibited in mice treated with the soluble IL-13 receptor. IL-13 administered to mice in the absence of allergen challenge still induced AHR, showing a causal role for IL-13 in this asthma model. Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro. Fisher JE, Rogers MJ, Halasy JM, Luckman SP, Hughes DE, Masarachia PJ, Wesolowski G, Russell RGG, Rodan GA, Reszka AA: Proc Natl Acad Sci USA 1999, 96:133-138. • Significance: Alendronate (ALN), a bisphosphonate (BP), is used clinically to prevent osteoporosis. While ALN and other BPs inhibit osteoclast activity, their precise molecular target(s) are unknown. Thus, the unraveling of their mechanism of action, begun in this paper, will aid in the selection of new drug targets and better guide the discovery of treatments for osteoporosis. Findings: Earlier experiments had shown that ALN inhibits cholesterol biosynthesis, and that exogeneous addition of mevalonate, farnesyl diphosphate (FPP) and geranylgeranyl diphosphate (GGPP) can reverse the blockade. To sort out which step of the mevalonate pathway is the key target of ALN, a more complete set of pathway intermediates were tested for their ability to reverse the effects of ALN in osteoclasts. Geranylgeraniol (GGOH) proved to be the most effective antagonist of ALN inhibition. ALN-treated osteoclasts show a seven fold increase in the activity of a 34 kDa kinase compared to untreated controls. This activation is blocked by GGOH. Note that farnesol and squalene are without effect. Thus, the authors propose that ALN inhibits a GGPP-forming step, which probably affects the prenylation by GGPP of key regulatory proteins in the cell.