Perspectives on immobilized proteins

Perspectives on immobilized proteins Staffan Birnbaum and Klaus Mosbach U n i v e r s i t y of Lund, Lund, S w e d e n New immobilization procedures, new and/or improved concepts and applications, and new methods in the characterization of immobilized proteins, are prominent among recent developments in the use of immobilized proteins in analysis. Current Opinion in Biotechnology 1991, 2:44-51

Introduction

Immobilization procedures

Protein immobilization, either by adsorption, covalent coupling and/or physical confinement, facilitates separation of that protein, as well as its specific binding parmer from the bulk phase. Thus, immobilization of proteins is often a prerequisite for success in many analytical as well as preparative applications in biotechnology. The ubiquitous use of this procedure is demonstrated by its extensive inclusion in the sections on biosensors, immunoassays, flow injection analysis and alFmity chromatography and, to a lesser degree, in other reviews in this issue. In this review, we have focused on three topics which we feel to be of primary importance: novel techniques in immobilization methodology, new and/or improved concepts and applications for analysis, and new methods in the characterization of immobilized proteins. Finally, we cite some illustrative examples of relevant techniques that have not been discussed in any other review.

The ultimate goal of the biochemical engineer who is immobilizing a protein is to obtain a preparation with optimal characteristics, such as 100% immobilization efficiency, 100% activity yield and enhanced stability. Developments in the immobilization procedure have focused on three main areas: the carrier (support or matrix), the immobilization procedure per se, or the protein itself.

Because we have tried to minimize the degree of overlap between this review and the others which mention immobilized proteins, we have not included publications which have been discussed elsewhere. We do hope, however, that readers will also scrutinize these other reviews in their search for noteworthy developments and applications of immobilized proteins. Furthermore, some of the publications which we discuss do not describe analytical applications of the immobilized protein, but we feel that the work reported is novel enough to be included. Lastly, we would like to refer the reader to three recent volumes in the Methods of Enzymology series which cover the field of immobilized enzymes and cells [1-3]. Of particular interest is the last volume [3] which focuses on analysis.

The carrier One of the primary concerns of the 'separation scientist' is the optimization of transport efficiency between the stationary and mobile phase. Recently, Regnier and co-workers [4 °,] reported the development of macroporous high-performance liquid chromatographic (HPLC) separation media in which the surface area of the material is increased by the presence of an interstitial network of smaller pores (500-15003,) between the larger throughpores (6000--8000/~). The so-called 'perfusion chromatography' material is characterized by the fact that intra-particle solute transport is strongly coupled to mobile phase velocity. Thus, band spreading, resolution of proteins and dynamic loading capacity remain unaffected when the mobile phase velocity increases up to several thousand centimeters per hour. Due to the larger throughpores, diffusional path lengths are minimized and the carrier exhibits transport characteristics of much smaller particles (1 I~m), but with a fraction of the pressure drop normally observed. The capacity and resolution of the material for a given amount of protein are equivalent to conventional high or low performance chromatographic media at a mobile phase velocity of 10 to 100 times greater than that usually employed. This paper [4"°] demonstrates the decreased separation time re-

Abbreviations BSA--bovine serum albumin; CliO---Chinese hamster ovary; CZE~capillary zone electrophoresis; HPLAC--high-perforrnance liquid affinity chromatography; HPLC--high-performance liquid chromatography; II--interleukin; RAC--receptor affinity chromatography; TIRF--total internal reflection fluorescence.

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Perspectives on immobilized proteins Birnbaum and Mosbach quired for analytical as well as preparative protein separations using flow-through ion exchanges and reversedphase particles. Evidently, this matrix will also be applicable to analysis with immobilized proteins. Other macroporous supports have been developed, and a patent has been issued recently [P1.].

Methods of immobilization The general method of immobilizing a protein to a support involves covalent coupling. Numerous methods exist and have been reviewed [5]. A recent report by Coleman et al. [6-] describes the preparation and use of co-polymer beads composed of vinyl dimethyl azlactone and methylene-bis-acrylamide which were shown to bind up to 400 mg of protein A per gram of cartier. The azlactone reacts with various nucleophiles (i.e. -NH2, - S H and - O H ) via a ring-opening addition. Reactions which involve primary amines on the protein, and subsequent rearrangements result in the coupling of proteins via amide bonds. This coupling reaction is fast (complete within 1 h) and efficient (up to 100% immobilization yield), and the preparations obtained are suitable for operation in the high performance mode. Whether this method is superior to others remains to be shown. An alternative immobilization method involves entrapment of the enzyme within a polymeric network. Rucka and Turkiewicz [7"] have described the direct incorporation of lipase into the interior of a polyvinyl chloride ultrafiltration membrane during the phase inversion stage of manufacturing. The membrane-entrapped lipase was reported to exhibit an apparent higher specific activity than the comparable soluble enzyme, as well as enhanced stability compared with the same enzyme immobilized through adsorption to the precast membrane. It is possible that the hydrophobic cartier imparts favorable substrate and product partitioning which is manifested as an increase in the specific activity of the immobilized lipase.

The protein One further aspect to be considered is the directed immobilization of the protein, which involves modifying it with either chemical or genetic engineering techniques. Kobos et aL [8"] have described a novel fluorocarbonbased immobilization technique. The protein is controllably modified with a perfluoroalkylating agent using conventional protein modification chemistry with, for example, imidazolides, isocyanates or N-hydroxysuccinimide esters, which react with amino residues of the protein. In general, 70% or more of the enzyme activity is retained after modification. The perfluoroalky!ated proteins can then be adsorbed onto a fluorocarbon support where they can play various roles, such as in affinity chromatography, bisensors or clinical diagnostics. Fluorocarbon supports have numerous advantages: they are chemically inert to acids, bases and organic solvents,

which allows stringent cleaning procedures to be used; they are mechanically stable and so can be used in the high performance mode; and they have a high specific gravity (1.8-2.1) which allows rapid separation by simple sedimentation. The support also exhibits minimal non-specific binding which lowers the detection limit in analytical applications. This method is useful for the immobilization of enzymes on gas-permeable fluorocarbon membranes. This has been demonstrated for the preparation of a urea electrode [9]. In some cases, significant activity loss may occur during adsorption of the modified protein onto the fluorocarbon support. To alleviate this, a hydrophilic spacer can be added between the perfluoroalkylating agent and the protein. For example, Lowe and coworkers [10] have used potyvinyt alcohol to link dyes to fluorocarbon supports which were later employed for protein purification. An alternative method to the chemical modification of proteins involves genetic engineering techniques. Thus, genetic fusion between the gene coding for a protein of interest with the gene coding for a polypeptide with high affinity for a binding partner can be efficiently immobilized (as well as purified), with 100% activity retention [11]. For further information on the use of genetic fusion in analytical biotechnology see the reviews by Scouten (this issue, pp 37-43), Brodelius (this issue pp 23-29) and Ngo (this issue, pp102-109). It has been shown [12-] that, using site-directed mutagenesis, it is possible to introduce amino acid residues into proteins that enable them to be immobilized at these sites. In this instance, a cysteine residue was introduced into glucose dehydrogenase and used to immobilize the protein to thiopropyl-Sepharose. This method, however, requires prior knowledge of regions of the protein that are exposed and that are not essential for catalytic activity. Incidentally, this work was initiated in an effort to link an NAD(H) analogue to the cysteine in order to obtain a catalytically active enzyme-coenzyme complex [13].

New or improved concepts and applications In the following section we will discuss the articles that convey some of the most important analytical applications of immobilized proteins. In some cases, the examples chosen demonstrate significant improvements in established methods (i.e. peptide mapping), or new applications (i.e. topographic mapping and structural studies). We have also chosen new examples of already established techniques (i.e. reversed affinity chromatography and high-performance liquid affinity chromatography, HPLAC). We will also refer to methods that have already been shown to operate in soluble systems and have now been demonstrated to function efficiently in immobilized systems (i.e. catalytic antibodies and cellfree translation). I.asdy, we will discuss examples of synthetic peptide and polymer preparations for the design of specific binding matrices.

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Analytical biotechnology

Peptide mapping Chemical or enzymatic cleavage of a protein followed by electrophoretic or chromatographic separation of the resuiting peptides represents a 'classic', yet still powerful, analytical procedure for protein identification and characterization. For example, peptide mapping is currently employed in quality control and monitoring of geneticallyengineered protein products [14]. However, for proteins in low abundance, the traditional soluble trypsin cleavage method of obtaining peptides is often incomplete and irreproducible because of the extreme dilution of trypsin required. If the trypsin-to-protein ratio is increased, the peptide product becomes contaminated with autolytic trypsin products. In order to alleviate this problem, Cobb and Novotny [15 o] used immobilized trypsin to achieve the complete and reproducible digestion of small amounts (50ng) of protein without contamination. A small amount of this peptide mixture (2 ng) was separated into fragments by capillary zone electrophoresis (CZE). In, five separate experiments, the standard deviation for all fourteen peaks obtained from CZE was less than 0.7%. Thus, immobilized trypsin in conjunction with CZE represents a highly sensitive and reproducible method for peptide mapping.

Topographic mapping Hacques et al. [16o] have reported the application of an immobilized protease for the study of chromatin structure. After limited proteolysis of the histone proteins with immobilized subtilisin, the accessible protein regions on the chromatin surface could be identified using antibodies specific for histones or histone proteins. Immobilized protease allowed for extent of digestion to be controlled easily, thus permitting site accessibility for subsequent sequential studies. Biophysical measurements on the selectively digested chromatin revealed structural changes that allowed the authors to postulate new roles for these particular histone tail regions in the adoption of condensed structures by chromatin. Unexpectedly, the chromatin decondensation brought about by the digestion, was not accompanied by the appearance of naked DN_A.Thus, the accessible protein regions identified appear to be involved in protein-protein interactions rather than protein-DNA interactions. This suggests that histone--histone interactions are crucial for stabilizing the chromatin structure.

Reversed affinity chromatography An alternative form of affinity chromatography is 'reversed affinity chromatography' in which a protein is immobilized to a stationary phase and used for both ligand and chiral molecule separations. Immobilized bovine serum albumin (BSA) and immobilized Col-acidglycoprotein are two examples of chiral stationary phases which have been used for the enantiomeric separation of numerous compounds [17,18]. One of the limitations of reversed atfinity chromatography is the low capacity of the sorbent caused by the large size of the protein molecule relative to its ligand. In an attempt to alleviate this prob-

lem, Erlandsson and Nilsson [19 °] have employed a proteolytic fragment (38 kD) of BSA for the enantiomeric separation of benzoin, oxazepam and morpholep. Although the enantiomeric separation was equal to or better than that achieved using intact BSA, the retention was not increased. This was probably due to the loss of nonselective binding sites caused by cleavage. The use of a smaller protein fragment has been demonstrated with the antibody-antigen interaction [20o.] (see below). During the past year, a number of other proteins have been employed for ligand separations. For example, immobilized cellulase has been shown to be a valuable chiral stationary phase for the separation of 13-adrenergic antagonists [21o]. In addition, immobilized proteases, such as chymotrypsin [22°] and trypsin [23 °] have been used for the separation of amino acids, their derivatives and peptides. In the case of chymotrypsin, enantiomeric separation of amino acids and amino acid derivatives was obtained. The ligand substrate was actually hydrolyzed so that separation occurred between the product formed and the remaining D-enantiomer. In the case of trypsin, the catalytic serine was modified to form the inert dehydroalanine so that no catalysis could occur. Immobilized anhydro-trypsin could then be used to separate peptides with terminal Arg or Lys residues. These applications have all been studied in the high performance mode and thus represent additional applications of HPLAC [24].

Receptor affinity chromatography In cases when high specificity and sensitivity are required, antibodies are an ideal choice for the separation of biomolecles, partly because of their high binding strength, ease of production and stability. For many substances, this is the only choice available as no other specific binding partner exists. For hormones such as cytokines, however, receptor affinity chromatography (RAC) offers a suitable alternative for their purification and analysis. For example, Weber and Bailon [25 °] have described the purification of recombinant intedeukin (IL)-2 by using its receptor, IL-2R, bound to a solid phase. The soluble form of the low affinity p55 subunit of the human IL-2 receptor (IL-2RANae) was produced by genetically engineered Chinese hamster ovary (CHO) cells and initially purified by IL-2 affinity chromatography. (The soluble receptor lacks the terminal 28 amino acids, which represent the transmembrane and cytoplasmic domains of the receptors, and yet contains the naturally N- and O-linked glycosylation sites). This receptor was coupled to a N-hydroxysuccinimide-activated silica support, and used to purify IL-2. Almost 1000-fold purification of CHO-produced recombinant IL-2 was obtained by RAC, and approximately 90% of the IL-2 load was bound. The specific activity of the IL-2 obtained after elution from the column was 1.8 x 107U/rag protein. This is twice the value obtained using a comparable immunoa/finity procedure based on monoclonal antibodies. The lower specific activity obtained from the immunoaffinity method was probably due to the fact that

Perspectives on immobilized proteins Birnbaurn and Mosbach the monoclon:d antibodies also recognized and bound oligomeric or aggregated forms of IL-2, which are considerably less active than the soluble monomeric form. Thus, various forms of IL-2, with varying degrees of biological activity, are obtained with monoclonal antibodies whereas essentially the monomeric, biologically fully active form of IL-2 is obtained with RAC. The authors also found that the receptor sorbent was stable for at least 500 runs. Conceivably, this system could be used for fermentation monitoring in recombinant hormone production. As more soluble receptors are being produced, this method is likely to increase in popularity for rapid hormone analysis. For a more in-depth review of contemporary receptor-based assay methodology see the review by Strosberg and Leysen (this issue, pp 30-36).

Catalytic antibodies The ability to produce catalytic antibodies (abzymes), first reported in 1986 [26,27] enables the creation of 'enzymes' with predisposed selectivity, provided an appropriate transition state analogue exists, or can be synthesized, for use as an immunogen. Last year, Blackburn et aL [28.] reported the use of catalytic antibodies in conjunction with a pH electrode for the analysis of phenytacetate. The use of catalytic antibodies as the molecular recognition element of a biosensor offers a number of advantages over conventional enzymes or immunosensors. Firstly, using catalytic antibodies, the range of potential substrate specificities is very much larger than that encountered using specific enzymes. Secondly, several methods are now well-established for the large-scale in vitro production of catalytic monoclonal antibodies, their modification through site-directed mutagenesis, and the production of the corresponding Fv fragments in Escbericbia colL Thirdly, antibodies are, to a large extent, conserved in structure (as opposed to enzymes which vary considerably). The conserved structure allows a standardized and optimized immobilization procedure to be developed which can be used for all analytes. Fourthly, antibodies are generally more stable than enzymes and, therefore, should have longer operational and storage life times. In particular, as the substrate molecule is catalyzed and the products diffuse away from the abzyme, it is selfregenerating (conventional immunosensors are generally limited with this respect). However, the abzyme sensor also has some major draw acks, which precludes its wide use as a biosensor. The sensitivity is relatively poor, in comparison with other immunosensors; thus for phenylacetate, the lower limit of detection (two times background noise) is 5 gM. In addition, the turnover rate is very slow (circa 1 rain -1). However, as the technique for preparing catalytic antibodies improves, these limitations are likely to be overcome. The recent demonstration that immobilized abzymes retain activity in organic solvents will broaden their field of application [29]. By and large, the use of enzymes in organic solvents is gaining increased attention [30]. In this context, the first reported attempts to use immobilized

enzymes for analysis in organic solvents should be mentioned [31o].

Cell.free translation Another topic that has been addressed for many years is that of cell-free gene expression. Until recently, these studies have been riddled with difficulties, stemming from the low yield and operational life time of the systems studied. However, Spirin and coworkers [32] have now reported a significant improvement in the amount of protein produced per mRNA molecule. This is achieved by immobilizing the translation system in an ultraffltration unit and operating the system continuously. Improvements to the original Spirin system in which transcription is coupled to the continuous translation system, have been reported [33"]. Consequently, it is now possible to produce and analyze expressed proteins that cannot be efficiently produced or obtained in an active form by in vivo expression systems, for example when the protein is rapidly degraded, is cytotoxic, or forms inclusion bodies. In addition, as post-translational modification is absent from this system, unmodified versions of the protein can be analyzed. It appears likely that this expression system will become a powerful analytical technique for studying the fidelity of protein synthesis, protein stability, folding and transport, as well as for the analysis of novel protein-engineered variants that could not be genetically expressed otherwise. Design of specific binding matrices Welling et al. [20-,] have reported the use of a synthetic peptide fragment as a ligand in immunoatfinity separations. As mentioned above (and also by Ngo, pp102-109), immunological recognition of an analyte is a powerful and widely used method of analysis. However, the harsh conditions required for regeneration of the immunosorbent, and the aspecific interaction of other regions of the immunoglobulin molecules, limit their use, particularly with respect to continuous or repeated assay methods and to sensitivity. In addition, the large size of the antibody limits the capacity of the sorbent for its antigen. These difficulties could be overcome by reducing the size of the molecule to a minimum. However, it is crucial that the biological activity (antigen binding site) and binding selectivity of the protein are retained. Welling et al. chose to test the well studied lysozyme-anti-lysozyme complex. A 13-residue antibody fragment with the largest number of contact residues, synthesized by solid-phase methodology and immobilized on Sepharose, was shown to separate lysozyme from a mixture of proteins as well as from calf serum. Lysozyme bound to the anti-lysozyme peptide adsorbent could be eluted by the addition of 0.25M sodium thiocyanate. This study clearly demonstrates that an immunoalfinity separation method with high selectivity is possible, using a 13-residue peptide ligand that mimics part of the antigen-binding site. Related to this method is 'paralog chromatography' [34] in which a representative set of paratope analogues (each synthesized from six amino acids which were randomly assem-

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Analyticalbiotechnology bled) are conjugated to a chromatographic adsorbent. This method was introduced as a mixed-mode general purification technique, but it remains to be seen whether the observed binding of the constructed paralog site really is specific and thus useful for analytical purposes.

Molecular imprinting An entirely different approach to produce tailor-made specific adsorption material and enzymic mimics, as well as biosensors, involves the technique of 'molecular imprinting' [35]. An imprint of the molecule of interest in a polymer is obtained by allowing polymerizable monomers to arrange themselves around this particular molecule before polymerization. After removal of the print molecule, the remaining cavity will recognize the original molecule by its shape and by the correctly positioned complementary groups of the polymer. Using this approach, excellent separations of racemic mixtures of amino acid derivatives into their optical antipodes have been obtained [36"]. The polymers can be likened to artificial antibodies. Following the same approach but using, for example, transition state analogues as print molecules, polymers expressing some catalytic activity have been obtained [37"]. Furthermore, the analysis of enantiomeric separations occurring in columns packed with 'imprinted gels' can be carried out continuously using potentiometric measurements [38"]. The selectivity of a protein, such as chymotrypsin, BSA or subtilisin, can be altered or markedly enhanced by either precipitation or lyophilization of the protein in the presence of the template molecule [39",40"]. This technique is known as 'bio-imprinting' and represents yet another method for preparing tailor-made binding parmers which may prove useful in future analytical and preparative applications.

Characterization of immobilized proteins Principles and methods for characterizing immobilized biocatalysts and affinity sorbents have been reviewed in many publications [1]. Here, we will discuss some new innovative procedures for immobilized protein characterization. Hoojimans et aL [41"] have described the use of an oxygen microsensor for the determination of the oxygen profile inside biocatalyst particles. This method allows the measurement of actual substrate concentration at different points and for different enzyme concentrations within the particle. Thus, one can determine whether the biocatalyst operates under diffusion limitation or substrate exhaustion. In addition, the external diffusion layer can be measured. For example, in their study on L-lactate-oxygen 2-oxidoreductase entrapped in agarose beads (of 2 mm radius), the extemal diffusion layer was approximately 50 l~n under the conditions used. Furthermore, internal diffusion limitation was observed as enzyme concentration increased. Measurements of this kind can be used to validate model predic-

tions obtained from separate determinations of various parameters. Fraaije et al. [42.] have described a method for determining the orientation of proteins adsorbed to a surface using total intemal reflection fluorescence (TIRF). The requirements for this method are that the protein of interest has a fluorescent group whose orientation relative to the rest of the protein is defined and constant, and that the protein does not change conformation upon adsorption. The authors were able to examine variations in the orientation of cytochrome c at an optically transparent SnO 2 film electrode, as a function of electrical potential at the interface. They found that protein orientation is affected by the interfacial potential during adsorption, but that once adsorbed, the protein orientation remains unaltered even as the potential is varied. Thus, this technique allows insight into the influence of electrostatic interaction during adsorption and can be useful in cases where specific orientation of certain molecules is desirable, such as for antibody adsorption at the surface of a biosensor.

Additional methods We would like to finish this review by mentioning some recent technical advances that have not been described elsewhere in this issue, but deserve recognition. Once again, these aitlnity techniques were developed, primarily, as preparative procedures but, in many instances, they have also been applied in analysis. Affinity precipitation is already renowned as a useful technique for the purification of oligomeric enzymes, such as dehydrogenases with bis-functionalized nucleotide derivatives [43 ]. More recently, Lilius eta/. [44" ] have described the use of protein engineering to add polyhistidine residues to galactose dehydrogenase, and the subsequent purification of the protein by metal atfinity precipitation. Atfinity partitioning in a two-aqueous phase system has also been successfully used for the purification and analysis of a number of enzymes [45,46]. By the addition of magnetic material to the system, the separation time can be lowered dramatically in the presence of a magnetic field [47"]. A number of firms now market various affinity membranes and hollow-fiber systems for both analytical and preparative purposes. Kinetic comparisons between conventional affinity chromatography material and affinity membranes for protein purification have recently been carried out [48"]. Lastly, we would like to refer to the recently introduced concept of weak affinity chromatography [49"]. This method exploits weak interactive forces (Kdiss = 1 - 1 0 - 4 M) and high binding site concentrations to obtain fast isocratic and dynamic separations with high performance, and selectivity. Such cooperative interaction chromatography might be applied in cases where

Perspectives on immobilized proteins B i r n b a u m a n d M o s b a c h mild elution procedures are required for product recovery.

ropolymer Support and its Application in AtFlnity Chromatography. J Chromatogr 1990, 510:177-187. 11.

References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest o• of outstanding interest 1.

MOSBACHK: Immobilized enzymes and cells: Part B. Methods Enzymol 1987, 135.

2.

MOSBACHK: Immobilized enzymes and cells: Part C. Methods Enzymol 1987, 136.

3.

MOSBACHK: Immobilized enzymes and cells: Part D. Methods Enzymol 1988, 137.

4. ••

AFEYANNB, GORDON NF, MAZSAROFF I, VARADY L, FULTON SP, YANG YB, REGNIER FE: Flow-through Particles for the High-performance Liquid Chromatographic Separation of Biomolecules: Perfusion Chromatography. J Chromatogr 1990, 519:1-29. A description of a new matrix which contains macroporous throughpores with interstitial smaller pores, allowing chromatography to be operated with reduced mass transfer resistance, but without a reduction in adsorbent capacity or an increased pressure drop. This results in exceedingly high flow rates. The theoretical background and several analytical and preparative examples are given. 5.

SCOUTEN WH: A Survey of Enzyme Coupling Techniques. Methods Enzymol 1987, 135:30-65.

6.

COLEMANPL, WALKER MM, MIIBRATH DS, STAUFFER DM, RASMUSSENJK, KREPSKI LR, HE1LMANNSM: Immobilization of Protein A at High Density on Azlactone-functional Polymeric beads and their use in Affinity Chromatography. J CJ~omatogr 1990, 512:345-363. The preparation of a new pre-activated matrix which can be operated at high flow rates and involves a new covalent coupling chemistry to nucleophilic ligands (i.e. proteins) is described. The coupling reaction is fast and was used to immobilize protein A at high density (400 mg/g). •

7.

RUCKAM, TURKIEWICZB: Ultrafiltration Membranes as Carri-

ers for Lipase Immobilization. Enzyme Microb Technol 1990, 12:52-55. Lipase from Rhizopus was incorporated directly into the interior of polyvinyt chloride ultraffltration membrane. The preparation exhibited improved activity and stability compared with the soluble or adsorbed enzyme.

12. .

PERSSONM, Bt3LOW L, MOSBACH K: Purification and Sitespecific Immobilization of Genetically Engineered Glucose Dehydrogenase on Thiopropyl-Sepharose. FEBS Lett 1990, 270:41-44. Glucose dehydrogenase was modified by site-directed mutagenesis to introduce a cysteine at residue 44 as an 'alFlnity tag' on the surface of the protein. Subsequently, the affinity tag was used for purification and site-specific immobilization of the protein to thiopropyLSepharose. 13.

PERSSON M, MANSSONMO, Bt3LOW L, MOSBACHK: Continuous Regeneration o f NAD(H) Covalently Bound to a Cysteine Residue Obtained by Site-directed Mutagenesis of Glucose Dehydrogenase. Biotechnology 1991, in press.

14.

GARNICKRL, SOILINJ, PAPAPA: The Role of Quality Control in Biotechnology: an Analytical Perspective. Anal Chem 1988, 60:2546-2557.

15. •

COBBKA, NOVOTNYM: High-sensitivity Peptide Mapping by Capillary Zone Electrophoresis and Microcolumn Liquid Chromatography, Using ImmobiliTed Trypsin for Protein Digestion. Anal Chem 1989, 61:2226-2231. 50ng of casein was digested with immobilized trypsin, and 2 ng of the resulting peptide mixture was used to obtain reproducible peptide maps with CZE. 16.

HACQUESM-F, MULLER S, DE MURCIA G, VAN REGENMORTEL MHV, MARION C: Use of all Immobilized Enzyme and Specific Antibodies to Analyse the Accessibility and Role of Histone Tails in Chromatin Structure. Bit~.hem Biophys Res Commun 1990, 168:637-643. Protease immobilization allows the controlled digestion of a target protein, thereby permitting the sequential investigation of the order of site accessibility, in this case for histones. Subsequent biophysical measurements gave insight into structure stability with regard to chromatin conformation. •

17.

ALLENMARKS: Optical Resolution by Liquid Chromatography on Immobilized Bovine Serum Albumin. J Liq Chromatogr 1986, 9:425--442.

18.

HERMANSSONJ: Enantiomeric Separation of Drugs and Related Compounds Based on their Interaction with Ctl-ACid glycoprotein. Trends Anal Chem 1989, 8:251-259

•

KOBOSRK, EVELEIGHJW, ARENTZENR: A Novel Fluorocarbonbased Immobilization Technology. Trends Biotechnol 1989, 7:101-105. Proteins can be modified with perfluoroalkylating agents and subsequently adsorbed to fluorocarbon supports. Fluorocarbon supports exhibit significant operational advantages which can be applied to affinity chromatography, biosensors and diagnostics. Alternatively, the protein may be linked via a spacer to the pertiuoroalkytating agent already adsorbed to the support. This helps to prevent activity loss. 8.

•

9.

KOBOS RK, EVELEIGH JW, STEPLER ML, HALEY BJ, PAPA SL: Fluorocarbon-based Immobilization Method for Preparation of Enzyme Electrodes. Anal Chem 1988, 60:1996-1998.

10.

STEWART DJ, PURVlS DR, LOWE CR: Atiinity Chromatography on Novel Perfluorocarbon Supports: Immobilization of C.I. Reactive Blue 2 on a Polyvinyl Alcohol-coated Perltuo-

UHLI~NM, MOKS T: Gene Fusions for Purpose of Expression: an Introduction. Methods Enzymol 1990, 185:129-143.

19. •

ERIANDSSONP, NILSSON S: Use of Fragment of Bovine Serum Albumin as a Chiral Stationary Phase in Liquid Chromatography. J Cbromatogr 1989, 482:35-51. The results of this report indicate that the use of protein fragments as chiral stationary phases in chromatography will lead to improved resolution (although some of the specificity is lost). 20. ))

WELL1NG GW, GUERTS T, VAN GORKUM J, DAMHOF R/~ DRIJFHOUTJW, BLOEMIIOFF W, WELILNG-WESTERS: Synthetic Antibody Fragment as Ligand in Immunoaffmity Chromatography. J Chromatogr 1990, 512:337-343. The 13-residue peptide, similar to part of the hypervariable segment of a monoclonal antibody directed against lysozyme, was coupled to Sepharose and used to purify lysozyme from serum. Sorbent-bound lysozyme could be eluted with a relatively low concentration of thiocyanate. This paper clearly demonstrates that synthetic peptide ligands which mimic antigen-binding sites can be employed for selective separation of proteins. 21. •

ERLANDSSONP, MARLEI, HANSSONL, ISAKSSONR, P E T I E ~ N C, PETYERSSONG: Immobilized Cellulase (CBH I) as a Chiral

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Analytical biotechnolosy Stationary Phase for Direct Resolution of Enantiomers. J Am Chem Soc 1990, 112:4573-4574. A demonstration that proteins other than mammalian transport proteins can be used for chiral separation (~-adrenergic antagonists, in this example). 22. .

JADAUDP, THELOHAN S, SCHONBAUMGR, WAINERIW: The Stereochemical Resolution o f Enantiomeric Free and Derivatized Amino A d d s Using an HPLC Chiral Stationary Phase Based on Immobilized (x-Chymotrypsin. ChiralitF 1989, 1:38--44. Silica-bound ct-chymotrypsin separates the enantiomers of several aliphatic and aromatic amino acids and their derivatives. Separation occurs as a result of either solute structure or enzyme activity. 23.

OHTAT, INOUE T, FUkq3MOTOY, TAKITANIS: Preparation of Aulaydrotrypsin-immobilized Diol Silica as a Selective Adsot-bent for High-performance AITlnity Chromatography of Peptides Containing Ar~'nitle or Lysine at their C-termini. Chromatograph/a 1990, 30:410-413. The catalytic setine of trypsin was converted to dehydroalanine to form the inert derivative anhydrotrypsin. When coupled to tresyl-activated silica, this sorbent could be employed for the rapid separation of Arg and Lys terminal peptides.

33. ••

BARANOVVI, MOROZOV IY, ORTLEPP SA, SPIRIN AS: Gene Expression in a Cell-free System on the Preparative Scale. Gene 1989, 84:463-466. 13-1actamase or dihydrofolate reductase-encoding plasmid and S-30 extract (transcription-translation system) from E. co/i were retained in a 1 ml ultrafiltration unit. Feed solution, containing nucleotides and amino acids, was continuously added and the polypeptide product continuously removed. A total of 200 ixg polypeptide was obtained in 50 h continuous operation. This is 50 times the amount obtained in a comparable static system. 34.

KAUVERLM, CHEUNG PYK, GOMER RH, FLEISCHERAA: Paralog Chromatography. Biotechniques 1990, 8:204-209.

35.

EKBERG B, MOSBACH K: Molecular Imprinting: a Technique for Producing Specific Separation Materials. Trends Biotec~ nol 1989, 7:92-96.

•

24.

OHLSON S, HANSSONIo GLADM, MOSBACHK, LARSSONPO: High Performance Liquid AITmity Chromatography: A New Tool in Biotechnology. Trends Biotechnol 1989, 7:179-186.

25. •

WEBERDV, BAILONP: Application of Receptor-AtTmity Chromatography to Bioafl'mity Purification. J Chromatogr 1990, 510:59-69. The IL-2 receptor, recombinantly produced in CHO cells, was covalently attached to silica and employed for IL-2 separation. The receptor-purified material had a higher specific activity than the monoclonal antibodypurified material. 26.

TRAMONTANOA, JANDA KD, LERNERRA: Catalytic Antibodies. Science 1986, 234:1566-1570.

36. •

ANDERSSONLI, O'SHANNESSYnJ, MOSBACHK: Molecular Recognition in Synthetic Polymers: Preparation of Chiral Stationary Phases by Molecular Imprinting of Amino Acid Amides. J Chromatogr 1990, 513:167-179. Molecular imprints of a number of L-amino acid aromatic amide derivatives were made in methacrylate-based polymer particles. These particles, when operated in the HPLC mode, exhibited efficient enantiomeric resolution of a racemic mixture of the amino acid amide used as the prim molecule and, in many instances, resolved amino acid amides other than the print molecule. 37. •

ROBINSON DK, MOSBACH K: Molecular Imprinting of a Transition State Analogue leads to a Polymer Exhibiting Esterolytic Activity. J Ooem Soc Chem Commun 1989, 14:969-970. The transition state analogue, pnitrophenyl methylphosphonate, was imprinted in poly[4(5)-vinylimidazole] cross-linked with 1,4-dibromobutane. The resulting polymer exhibited an enhanced rate of pnitrophenytacetate hydrolysis. 38.

ANDERSSONLI, MIYABAYASHIA, O'SHANNESSEY nJ, MOSBACH K: Enantiomeric Resolution of Amino Acid Derivatives on Molecularly Imprinted Polymers as Monitored by Potentiometric Measurements. J ~romatogr 1990, 516:323--331. A flow-through potentiometric column packed with synthetic polymers imprinted against L-phenylalanine anilide was employed to follow the enantiomeric separation of phenylalanine anilide. •

27.

POLLACKSJ, JACOBSJW, SCHULTZPG: selective Chemical Catalysts by an Antibody. Science 1986, 234:1570-1573.

28.

BLACKBURNGF, TAtIEY DB, BOOTH PM, DURFOR CN, MARTIN MT, NAPPERAD, REESAR: Potentiometric Biosensor Employhag Catalytic Antibodies as a Molecular Recognition Element. Anal O0em 1990, 62:2211-2216. This is the first report of the use of catalytic antibodies as the molecule recognition element on a biosensor. The catalytic property of the enzyme not only produces the signal, but also continually regenerates itself, thereby alleviating one of the recurring difficulties associated with immunosensors. •

29.

JANDA KD, ASHLEYJA, JONES TM, MCLEOD DA, SCHLOEDER DM, WEINHOUSE MI2 Immobilized Catalytic Antibodies in Aqueous and Organic Solvents. J Am Chem Soc 1990, 112:8886-8888.

30.

KLmANOVAM: Enzymes that Work in Organic Solvents. O0emtech 1986, 16:354-359.

31. •

STASINSKAB, DANIELSSON B, MOSBACH K: The use of Biosensots in Bioorganic Synthesis: Peptide Synthesis by Immobilized at.Cbymotrypsin Assessed with an Enzyme Thermistor. Biotec]snol Techniques 1989, 3:281-288. Analysis of peptide synthesis in organic solvents using an enzyme thermistor with immobilized ~t-chymotrypsin is described. 32.

SPIRIN AS, BARANOVVI, RYABOVALA, OVODOV SY, ALAKHOV YB: A Continuous Cell-free Translation System Capable of Producing Polypeptides in High Yield. Science 1988, 242:1162-1164.

39. •

STAI-ILS, MANSSON MO, MOSBACH K: The Synthesis of a 0amino Acid Ester in an Organic Media with ct-ChymottT~in Modified by a Bio-imprinting Procedure. Biotechnol Lett 1990, 12:161-166. The enzyme-inhibitor complex, chymotrypsin and N-acetyl-D-tryptophane, was precipitated in 1-propanol. The precipitate could then be used to sythesize N-acetyl-n-ttyptophan ethyl ester in cyclohexane. 40. •

BRACO L, DABUUS K, KLmANOVAM: Production of Abiotic Receptor by Molecular Imprinting of Proteins. Proc Natl Acad Sci USA 1990, 87:274-277. When BSA was mixed with a 'print' molecule, such as L-tartaric acid or phydroxybenzoic acid, in an acetone-water solution and lypholized, enhanced binding of the print molecule to the protein was subsequently observed both in organic solvent, and when immobilized to controlled pore glass. 41. •

HOOIJMANSCM, RAS C, LUYBENKCAM: Determination of Oxygen Profiles in Biocatalyst Particles by means o f Combined Polarographic Oxygen Microsensor. Enzyme Microb Technol 1990, 12:178-183. The substrate profile within the biocatalyst particle (agarose containing L-lactate 2-monooxygenase) was measured with an oxygen microsensot. This allowed diffusion limitation at various enzyme concentrations, as well as substrate exhaustion to be determined. 42. •

FRAAIJEJGEM, KLEIJNJM, VAN DER GRAm M, DIJT JC: Orientation of Adsorbed Cytochrome c as a Function of the Elec-

Perspectives on immobilized proteins Birnbaum and Mosbach trical Potential o f the Interface Studied by Total Internal Reflection Fluorescence. BioptyysJ 1990, 57:965-975. A method for determining the orientation of proteins adsorbed on a surface is described. Proteins which contain an intrinsic fluorescent label such as porphyrin in cytochrome c, can be studied by TIRF based on the ratio of light intensities at two polarization modes. 43.

LARSSONPO, FLYGARES, MOSBACH K: Attinity Precipitation.

Methods Enzymol 1984, 104:364-369. 44. •

IaLR2SG, PERSSONM, BLOW L, MOSBACHK: Metal Affinity Precipitation o f Proteins Carrying Genetically Attached Polyhistidine Affinity Tails. Eur J Biochem 1991, in press. A DNA segment encoding five histidine residues was fused via genetic engineering to the y-terminal of galactose dehydrogenase. This 'affinity tail' could be exploited to purify the modified enzyme by either affinity precipitation or alBrtity chromatography, using metal chelators. The polyhistidine tail could then be removed with carboxypeptidase A. 45.

JOHANSSONG: Affinity Partitioning of Enzymes. In Separations Using Aqueous Phase Systems: Applications in Cell Biology and Biotechnology edited by Fisher D, Sutherland IA. New York:

in a magnetic field. The time taken for separation to be achieved was decreased by up to 240 000 tmaes. 48. •

UNARSKAM, DAVIESPA, ESNOUF MP, BELLHOUSEBJ: Comparative Study of Reaction Kinetics in Membrane and Agarose Bead Affinity Systems. J Ctyromatogr 1990, 519:53-67. Reaction kinetics between y-globulin and immobilized protein A are much more favourable when microporous nylon membrane is used as a support than with conventional agarose bead affinity supports because the solution is forced through the membrane support. 49. ZOPF D, OHLSON S: W e a k - a l ~ t y Chromatography. Nature • 1990, 346:87-88. A new method using readily reversible biospecific recognition as a basis for chromatographic separation is described. The analytical, as well as preparative, applications are discussed.

Annotated Patents • ••

of interest of outstanding interest

Plenum, 1989, pp 7-14. 46.

A L B E ~ N P-A (ed): Particles and Macromolecule~ New York: John Wiley, 1986.

FLYGARES, WIKSTROMP, JOHANSSONG, LARSSONPO: Magnetic Aqueous Two-phase Separation in Preparative Applications. Enzym Microb Technol 1990, 12:95-103. Addition of magnetically susceptible material to an aqueous two-phase system induces rapid phase separation when the mixed system is placed

P1. MILSSONKG, MO~3BACHK: Macroporous Particles for Cell Cul• tivation and Chromatography. 1990, US Pat No 4 935 365. A method for the preparation of macroporous polymers that are applicable both as microcarriers and in chromatography is described.

47. •

S Bimbaum and K Mosbach, Pure and Applied Biochemistry, Chemical Center, University of Lund, Lund, S-22100 Sweden.

51