Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coli

Biochimie (1991) 73, 647-655 © Soci6t6 franCaise de biochimie et biologie mol6culaire / Elsevier, Paris

647

Genetic engineering and overexpression of ribosomal L12 protein genes from three different archaebacteria in E coil AKE K6pkel,2*, F Hannemannl, T Boeckhl tMax-P lanck-lnstitut fiir Molekulare Genetik, Abteilung Wittmann, lhnestrasse 73, D- I O00 Berlin 33, Germany; ZDepartment of Biochemistry and Microbiology, University of Victoria, PO Box 3055, Victoria, BC, Canada V8W 3P6

(Received 13 November 1990; accepted 12 March 1991)

Summary - - Genes coding for ribosomal protein L I2 from Methanococcus vanniefii (Mva), Halobacterium halobium (Hha) and Sulfolobus solfataricus (Sso) have been subcloned in the polylinker region of pUC 19. An efficient Shine-Dalgarno sequence has been attached to the 5' end of the genes, and two ochre stop codons have been created at their 3' ends, where necessary. In addition, mutants of the MvaL12 and HhaL12 genes were constructed, which coded for a cysteine residue at the C-terminus of the protein. The constructs were transferred together with the pUC 19 polylinker as gene cartridges into different expression vectors. These constructed plasmids were transformed in the appropriate E coli hosts and tested for expression. Two systems were found to work efficiently for overexpression, namely the pKK223-3 vector featuring a tac promoter, and the pT7-5 vector featuring a T7-promoter. The overexpressed proteins were purified to homogeneity; their purity was investigated by one and two-dimensional gel systems, amino acid analysis and N-terminal protein sequencing for 10 steps or more. The amount of protein purified from E coil test cultures bearing the expression plasmids was always more than 2.5 mg/l of medium used. overexpression / ribosomal proteins / gene modifications / protein purification

Introduction Gene expression and the resulting synthesis of cellular proteins on the ribosome are essential for all living cells; therefore ribosomes are ubiquitous. Although ribosomes maintain the same function in all organisms, it was found that ribosomes from eubacteria, archaebacteria and eukaryotes differ to a great extent in the sizes, numbers and sequences of their constituents [ 1]. In addition electron microscopy revealed different ribosome shapes in the different cell types [2, 3]. These findings largely contributed to the classification of the distinct groups of organisms and gave hints for the evolutionary process from the eukaryote to the modem organisms [4-6]. As expected, equivalent ribosomal proteins from the different kingdoms share similarities in their sequences. Interestingly, the degree of similarity within the different protein families (equivalent ribosomal proteins in different organisms) varies largely from

*Correspondence and reprints Abbreviations: DTE, dithioerythritol; DMF, dimethylformamide

one protein family to another. In general, the amino acid sequences of archabacterial ribosomal proteins were found to be more similar to those of eukaryotes than to those of eubacteria. However, for the most conserved protein families the degree of sequence similarity between the different kingdoms was more alike [6]. Multiple alignments of equivalent ribosomal proteins from different organisms show highly conserved residues, which are predictably important for special functions of the protein. Therefore, it was quite unexpected that the protein sequences of one of the functionally important protein domains of the ribosome, namely the stalk protein complex [7], were so poorly conserved ([6] for citations of the L12 protein sequences). The primary protein sequences of its constituents, the L10 and L I 2 proteins, differ so drastically between the eubacteria on one hand and archaebacteria and eukaryotes on the other hand, that no unambiguous alignment could be found [8, 9], despite their similar and unique amino acid composition. Furthermore, within the aligned group of archaebacterial and eukaryotic L I2 proteins and also among the archaebacterial kingdom itself the degree of conservation is rather low compared to other ribosomal protein families.

648

AKE K/ipke et al

The E coli L I 2 protein is the only ribosomal protein occurring as two dimers per ribosome, where depending on the growth rate two of the four copies are N-terminally acetylated L12 proteins called L7 [10]. It is supposed that one dimer constitutes the stalk protuberazlce, while the other dimer is located at the base of the stalk in the body of the 50S subunit of the ribosome [11, 12]. The same ratio of four L12 protein copies to one L10 protein per ribosome was found for the equivalent proteins in archaebacteria [13] and eukaryotes [14]. The LI2 protein is essential for the translational process. It interacts with the elongation factors and activates their GTPase activity. Both dimers can be easily isolated from most ribosomes, also as a complex together with the L10 protein (stalk protein complex), by simple salt/ethanol washes of the ribosomes. In E coli, protein L10 anchors the LI2 protein dimers in the large subunit of the ribosome by binding to the 23S rRNA and to protein L11. The 3-D structure of a C-terminal fragment of the E coli L12 protein was resolved by X-ray diffraction crystallization [15]. The orientation of the protein in the ribosome was examined utilizing antibodies against the Nand C-terminal part of the protein, respectively [ 16]. It was found that the N-terminal domain is located in the body of the ribosome, probably bound to the L10 protein, while the globular C-terminal domain remains free at the tip of the stalk for one dimer and near the base of the stalk for the second dimer ([17] for a E coli L12 review). Due to the differences in the amino acid sequences of the archaebacterial and eukaryotic L I2 group on one hand and the eubacterial group on the other hand, results obtained for the structure-function relationship of this protein in E coli cannot be implied to the archaebacterial and eukaryotic proteins directly. The knowledge that the L12 proteins constitute the stalk in the ribosomes of all kingdoms, combined with their great variety in primary structure and the known function of these proteins in E coli made them interesting for further investigations. Autoimmune antibodies of lupus patients bind to the highly conserved and hydrophilic C-termini of the LI2 and L10 proteins of humans. This fact indicates that these sequences are located at the surface of the ribosome and probably at the tip of the stalk protuberance. In electron micrographs these antibodies were found at the tip of the stalk protuberance of human 60S subunits (G Stifffler et al, personal communication). It may be suggested that the C-terminal sequence of the archaebacterial L I2 proteins are similarly exposed. This has led us to the strategy of modifying the C-terminus of archaebacterial L I2 proteins, to make the protein suitable for specific heavy atom labeling. Crystallized halobacterial ribosomes bearing distinct labeled mutant L12 proteins would facilitate phase evaluation in the X-ray

structure determination of these large particles [18]. Therefore cysteine residues as specific attachment groups were introduced as C-terminal amino acids in some L 12 protein mutants by genetic engineering. Large amounts of purified protein will be utilized for 2-D-NMR, crystallographic studies and functional tests, eg binding studies of these proteins to elongation factors. In addition, various mutants are required for the investigation of structure-function relat~onships in the L12 protein sequence. Therefore, overexpression of the archaebacterial L I2 proteins in E coli proved to be the appropriate way to obtain the desired amounts of purified proteins, in combination with fast and versatile access to protein mutants. Materials and Methods DNA handling and cloning procedures were performed according to Sambrook et al [19]. Separation of DNA fragments was partly done on HPLC as previously described [9]. Used E coli strains E coli XLI blue [20] has the followingchromosomal mutations:

reeAl, Alac, endAl, gyrA96, thi, hsdR17, supE44, relAl; and an F' with the mutations proAB, laclq, IacZAM15, Tnl0. E coli BL21 DE3 has the mutations P-, erupT, rB-ma- for the BL21 strain and the DE3 lysogen contains the minimum region of phage 21, a fragment of the lad gene, the lac UV5 promoter, beginning of iacZ gene and the gene for T7 RNA polymerase [21l. Purification oft he oligonucleotide linkers

The oligonucleotides, synthesized by an automated DNA synthesizer (Applied Biosystems 28013), were purified according to [22]. Annealing was performed with equimolar amounts of corresponding oligonucleotides in 10 mM Tris-HCl containing 50 mM NaCI, 1 mM dithioerythritol and 10 mM MgCI2. The mixture was boiled for one rain and slowly cooled down to room temperature. Approximately 500 fmol were used in the ligations with DNA fragments and the vector. Overexpression in test cultures

Test cultures (20 ml to 2 !) of the strains containing the expression vectors were grown at 37°C with vigorous shaking in LB medium containing the appropriate antibiotics. When the optical density reached A6so = 0.25 for pKK- and 1.0 for pT7vectors, isopropyl-D-thiogalactopyranoside(IPTG) was added to a final concentration of 0.5 mM and the incubation was continued for 4 h. Aliquots of the cells were taken at different times, the optical density was measured, the cells were collected by centrifugation and calculated amounts of SDS loading buffer were added (100 ~tl SDS loading buffer per 1 ml medium of cells at an optical density of A~so= 1.0). The cell pellets were resuspended, incubated at 50°C for 10 rain, boiled for 5 rain, chilled for 10 rain, centrifuged at 16 000 g for 10 min and the supernatant analysed on SDS acrylamide gels [23, 24l. After 4 h the remaining cells were harvested by centrifugation at 4 000 g, resuspended in TMA I buffer and lysed by sonification. The lysate was centrifuged at 15 000 g for 15 rain to precipitate the bacterial debris, followed by a centrifugation

Archaebacterial ribosomal L 12 proteins

649

at 252 000 g for 4 h or overnight to remove ribosomes and other suspended particles. The clear supematant was dialysed against water, lyophilized and purified by HPLC.

The clear supematant was dialysed against water for 16 h with frequent water changes and lyophilized. The pellet was stored at -20°C.

Overexpression in a fermentor

Separation of the overexpressed proteins fi'om the cytosolic protein fraction by DEAE ion exchange chromatography

A 25- and a 100-liter fermentor were used for growth of the bacteria. The medium was LB-medium containing half of the normal NaCI concentration. When the optical density reached the designated value (A26o = 0.25 for pKK- and A26 o - 1.0 for pT7-vectors) the culture was induced with IPTG (0.05 mM for pKK- and 0.1 mM for pT7-vectors). Four h after induction the cells were harvested, resuspended in TMA I (10 g cells per 15 ml buffer) and iysed in a french press. The lysate was centrifuged at 15 000 g for 15 min and at 252 000 g overnight.

The lyophilized cytosolic proteins prepared from the cells grown in the fermenter were dissolved in 20 mM TrisoHCi containing 30% DMF and 1 mM DTE (buffer A) and applied to a DE52 (Whatman) ion exchange column (5 x 70 cm) equilibrated with buffer A. Elution was performed using a linear gradient from 0 to 0.5 M KCI in buffer A for MvaLl2/ MvaLl 2cys and from 0 to 1.0 M KCI in buffer A for HhaLl 2/ HhaLl2cys (fig 1). Aiiquots of the fractions were analyzed by

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purity of the products was checked by 2D-gel electrophoresis (fig 3), using the first dimension according to Strum and Visentin [25] and the second dimension according to Laemmli [23]. All proteins were analyzed by amino acid analysis (Applied Biosystems 420 Derivatizer/130A Separation System or alternative ortho-phthaldialdehyde derivatization and analysis in a HPLC System of Waters/Millipore) and N-terminal protein sequencing (Knauer 810 Modular Sequencer or Applied Biosystems 475A). Optimization of the expression conditions for the pKK vectors

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Different incubation periods before induction and various concentrations of IPTG were applied to 100 ml cultures of pMvaLl2 in E coli XLI blue. Aliquots of the culture were taken, analyzed on SDS polyacrylamide gels, as mentioned above, and quantitated by a gel scanner at 530 nm using another cellular protein in the same lane as a control, The amount of MvaLl2 protein found was normalized by the amount of this reference protein, which seemed to be related to cell density rather than growth conditions. It was found that for the pKK223-3 vector containing the MvaLl2 gene the amount of overexpressed protein varied little as long as the induction was in the early log phase, while induction in the late log phase resulted in poor protein production (table I). The early induction at an optical density of A650 = 0,25 gave the highest amount of overexpressed protein. To reduce the cost of IPTG for larger medium volumes the concentration for the induction was varied. It was found that the IPTG concentration could be reduced to 1/10 (0.05 mM) with a loss of about 30% in the protein amount produced (table II).

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SDS-PAGE, and appropriate fractions were pooled, dialysed against 10 mM mercaptoethanol in water and lyophylized. Mercaptoethanol (10 raM) was present in all buffers utilized in the following procedure. The DE52 purified proteins from the fermentor preparation, or the crude cytosolic mixture for the analytical scale, were dissolved in HPLC buffer A and injected on a C4-reverse phase HPLC column (8 x 250 or 15 x 120 mm). Buffer A was 50 mM ammonium acetate, pH 7.0, buffer B was 2 propanol. Due to its higher hydrophilicity the halobacterial proteins eluted much earlier than the equivalent methanococcal proteins. For the overexpressed proteins the HPLC elution profile always ~howed a non-uniform peak (fig 2), The eluted fractions were scanned by SDS gels, the desired protein peak was pooled and rechromatographed where necessary. The fractions bearing the purified protein were lyophilized and redissolved in water. The

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Table I. Variation of the incubation periods before induction. The cells were grown to a certain optical density, induced with 0.5 mM IPTG and harvested after additional 4h. A~5o

Reference area

0.1 0.25 0.5 1.0

180 140 220 180

MvaL12e Crude Corrected area area

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260 280 260 100

%Max

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Optimization of the expression conditions for pT7 vectors The duration of overexpression, the time and the IPTG concentration for induction were also optimized for the pT7 vector system. 100 ml LBAmp medium were inoculated with ! ml overnight culture of E coli BL21-DE3 bearing the pT7-SsoLl2 vector and grown to an optical density of A6so = 0.5. The cells were induced with 0.5 mM IPTG and a sample was taken at 30 min, l, 2 and 4 h analyzed on SDS-PAGE as described above. The greatest amount of SsoLl2e was found after 4 h. Subsequently, 100 l.tl of the described overnight culture was added to 10 ml LBamp_medium and grown to optical densities of A650= 0.25, 0.5 and 1.0, respectively, induced with 0.5 mM IPTG and grown for 4 h. It was found that the amount of protein produced per cell was greater and the bacteria grew to a higher density, if the culture was induced late (at A6so= 1.0). A control experiment showed that this holds true for the plain pT7-5 vector in E coli BL21-DE3, where induction in the phase of exponential growth seemed to inhibit cell growth. The IPTG concentration for induction at a cell density of A6so = 1.0 was varied from 0.01, 0.05, 0. l to 0.5 mM. The amount of overexpressed protein after 4 h of expression was virtually the same for these various IPTG concentrations, when estimated from SDS-PAGE gels. As expected, the control experiment of uninduced cells showed no expression after 4 h of additional growth.

The M v a L l 2 gene was modified to code for a cysteine on the C-terminus o f the protein in order to provide a site for specific modification eg with heavy atom derivatives for X-ray crystallographic studies. T w o oligonucleotides were synthesized and annealed to constitute a linker. The M v a L l 2 gene from p M v a L l 2 was cut by E c o R l on its 5' end and by Heall on its 3' end. The resultant fragment was ligated with the

Table III. Amino acid analysis of purified MvaL12 e (Applied Biosystems 420 Derivatizer/130A Separation System) and MvaLl2cy~ (ortho-phthaldiaidehyde precolumn derivatization and analysis in a HPLC System of Waters/ Millipore). Lysozyme was used as a reference for the determination of cysteine residues. The values given were not corrected to compensate for partial hydrolysis or destruction of amino acids, nd = Not determined. Amino acid

MvaL l 2e Coded Found

6 !1 ! 5 3 1 33 2 2 10

6.5 1 !.0 1.1 4.3 3.7 1.3 30.5 2.0 1.7 10.5

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6.2 12.2 1.6 6.8 2.6 1.3 35.8 2.1 1.1 8.8

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(MvaL12 e) was tested for its biological activity and found able to replace the genuine halobacterial L I2 protein in halobacterial ribosomal 50S subunits [26]. M v a L 1 2 e, from a 100 1 culture, was purified to homogeneity. T h e amino acid analysis (table III) o f an aliquot was extrapolated to the a m o u n t o f pure protein present. Fifty m g o f purified M v a L 1 2 e protein could be obtained.

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Fig 4, Construction of the MvaLl2cy.~mutant: the MvaLl2 gene-cartridge was cut by Haell and ligated with the synthesized linker (shown in white) into pUC19. The oligonucleotide linker contained the altered DNA sequence to code for a cysteine at the 3' end of the gene. designed oligonucleotide linker into the EcoRI and HindllI digested pUC 19 vector (fig 4). Recombinant clones were checked by restriction enzyme analysis and DNA sequencing. The EcoRl, Hindlll fragment (MvaLl2 cys gene cartridge) of the resultant plasmid was subcloned into pKK223-3 and transformed in E coli XLI blue. The overexpressed protein was purified according to the procedure outlined above

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Fig 5. Construction of the HhaL12 gene-cartddge (top part) and the HhaLl2cy~mutant (bottom part of the figure): to introduce the Mva Shine-Dalgamo sequence (indicated by stars) into the halobacteriai gene, the Hhall2 gene was cut by Fokl and ligated with the designed linker (shown in white) into pUC 19. The oligonucleotide linker contained the SD-sequence as found for the MvaL12 gene in the same spacing to the ATG start codon (top). The fragment, resulting fro~, a StuI digest of the HhaL)2 genecartridge, was ligated with the oligonucleotide linker (shown in white), containing the new codon, into pUC 19 (bottom).

Archaebacterial ribosomal L I 2 proteins [27]. It was subcloned into pUC19 as a 433-bp AvaI fragment. After subcloning into pKK223-3 no overexpression was achieved. Therefore, the original halobacterial SD-sequence was replaced by the methanococcal SD-sequence,which already had proven to be functional for the overexpression of the MvaL12 protein in E coli. The HhaL12-gene cartridge (cut out of the plasmid by EcoRI and HindHI) was digested with FokI, purified by agarose gel electrophoresis and ligated with a designed oligonucleotide linker into pUC 19 (cut by EcoRI and HindIII). The linker contained a 5' EcoRI site, the methanococcal SD-sequence (in the same spacing to the ATO-start codon that was found to work for the MvaL12 gene), and a 3' FokI site (fig 5). The recombinant plasmid was checked by restriction enzyme digests and DNA-sequencing. The modified gene cartridge was subcloned into pKK2233 and checked by restriction enzyme digests. Overexpression of this plasmid in E coli XLI blue was obtained and the resulting protein was analyzed by amino acid analysis (table IV) and protein sequencing. The expressed protein had an amino acid sequence identical to the genuine protein from Halobacterium halobium and no N-terminal modifications. This is different from the overexpressed MvaL12 e protein which contained a N-terminal acetylation not present in the genuine Methanococcus vannielii protein [26]. Construction and overexpression of HhaLl 2 cys To obtain a HhaLl2 protein with a cysteine as the C-terminal residue, the HhaLl2 gene cartridge was digested with StuI and ligated with a new oligonucleotide linker featuring: (1) a 5' StuI site (blunt end); (2) a changed DNA sequence to code for a cysteine, two ochre stop codons at the C-terminus, and (3) a 3' Sau3aI site (fig 5). This construct was ligated into pUC19 and the recombinant clone was analyzed by restriction enzyme digests and DNA-sequencing. The modified gene cartridge was subcloned into pKK2233 to yield the expression plasmid. The protein was overexpressed and purified as described for HhaLl2. The purified HhaL12cys was not N-terminally blocked, had the predicted amino acid composition (table IV) and the correct N-terminal protein sequence of the HhaL 12 protein. Modification of this protein for crystallographic studies is currently under way. Subcloning modification and overexpression of the SsoLI2 gene The L12 from Sulfolobus solfataricus was cloned and sequenced as described [28]. The gene was subcloned into pUC 19 as a 1119 bp NsiI fragment. The recombinant clones were tested with a PstI, ltindIII double digest, where PstI cuts in the last third of the gene and

653

Table IV. Amino acid analysis of purified HhaL12e and HhaLl2cys by ortho-phthaldialdehyde precolumn derivatization. Lysozyme was used as a reference for the determination of cysteine residues. The values given were not corrected to compensate for partial hydrolysis or destruction of amino acids, nd= Not determined. Amino acid

HhaLl2 e Coded Found

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HindIII cuts in the polylinker of pUC 19. The plasmid that had the HindIII site 3' to the gene was cut by StyI, the overlapping ends were flied by T4-polymerase and the gene was released from the vector by an EcoRI digest. The resulting 700-bp fragment was ligated into EcoRI and Sinai site of pUC 19 again. The recombinant plasmid was cleaved with RsaI and HindIII, the resulting 39 l-bp fragment was purified by HPLC and ligated with the designed linker into the pUC19 vector (fig 6). The linker contained the MvaLl2 SD-sequence used for the other plotein genes. The reombinant clone was analyzed by resuiction enzyme digests and DNA-sequencing from both sides. The SsoL12 gene cartridge was then subcloned into pKK223-3. The recombinant clones showed very poor growth and were unstable after several overnight cultures. It was fqund that the gene expression was not controlled by the tac-operator/promotor; instead the protein was already present in large amounts before induction. This was unexpected since the E coil strain used (XLI blue) has a lac lq mutation in the promotor

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of the lac-repressor gene, which helps to maintain a high titer of lac-repressor molecules in the cytosol (even with the high copy number of the overexpression plasmid) and the same construct was successful for all other L I2 genes. To circumvent this gene related problem, the SsoLl2 gene cartridge was subcloned into pT7-5 [29], which contains a T7-promotor 5' to the gene and the 13-1actamase gene in a reversed orientation to the T7-promotor. The recombinant plasmids Table V. Amino acid analysis of purified SsoL 12e (Applied Biosystems 420A Derivatizer/130A Separation System). The values given were not corrected to compensate for partial hydrolysis or destruction of amino acids.

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Asp Glu Ser Gly His Arg Thr A!a Pro Tyr Val Met lie Leu Phe Lys

5 20 6 7 ! ! 4 18 3 2 7 2 7 9 1 12

5.0 19.3 5.0 6.5 1.0 1.2 3.6 18.0 1.9 2.0 7.0 1.1 6.8 9.6 1.1 12.9

were transformed into the E coli strain BL21-DE3, which contains a T7-polymerase gene under the control of a lac-promotor. These cells were able to grow normally prior to induction. Overproduction of the SsoL12 protein was achieved with even small concentrations (10 l.tM) of IPTG in the medium. It was found that for this system it is best to induce the cells in the late phase of exponential growth. This allows them to grow normally after induction and increased the amount of produced SsoLl2 e protein. The overexpressed protein was purified according to the procedure outlined for MvaL12. A big broad peak in the reverse phase chromatogram was the purified SsoL12 e protein. It was characterized by amino acid analysis (table V) and protein sequencing. Higher amounts of protein were calculated for the amino acid analysis and SDS-gel compared to results from the protein sequencing. A likely explanation is that a major portion of the protein is N-terminally blocked, as this is also known for the SsoLl2 protein isolated from Sulfolobus solfataricus and the overexpressed MvaLl2 e protein in E coll. The SsoLl2 e protein and its designed mutants are currently under investigation. Discussion

L12 proteins from three different archaebacteria, each representing a major branch of the archaebacterial kingdom, were overexpressed in E coll. The SDsequences 5' to some of the genes had to be modified to achieve the overexpression. Only the two overexpression systems described were found functional for the different genes. The gene to be overexpressed seemed to dictate the system which can be used successfully. Some constructions were lethal or unstable, while others showed no expression. The successful systems described in this paper yielded an expression level of about 2.5 mg overexpressed protein or more per liter of medium.

Archaebacterial ribosomal L l2 proteins

The proteins were purified by a fast and reliable procedure, where the final purification step is a preparative reverse phase HPLC separation. All investigated L12 proteins show a broad or double peak which show no differences in mobility on SDS-PAGE or in amino acid analysis composition. The L I2 proteins were renatured, as indicated by different mobility on SDS-PAGE gels before and after heat denaturation. It had previously been shown that the MvaLl 2 ~ protein is active as a ribosomal constituent [26]. The cloned protein genes, together with their succesful overexpression, give access to designed protein mutants. Overexpression of mutant proteins coding for a cysteine at their C-terminus was also obtained. These mutants are useful for the specific labeling of a distinct position in the protein L l2 with heavy atom derivatives, following the reconstitution into ribosomes, which is a necessary step to determine the phases in the X-ray diffraction patterns of ribosomal crystals. If the protein mutants, labeled at the cysteine with the bulky gold cluster, can be reconstituted into ribosomes, the orientation of the L I2 with the Cterminus at the outer tip of the stalk protuberance would be evident. This would indicate a similar relative orientation for the L l2 proteins as found in E coli, in contrast to the highly diverged primary sequence. Mutagenesis studies for the Sulfolobus solfataricus L l2 protein are in progress. With these mutant proteins it should be possible to investigate the function of different parts of the LI 2 protein sequence. The huge amounts of purified, overexpressed protein available enable biophysical investigations by 2D-NMR and crystallographic studies. These studies will allow an insight into the three-dimensional structure of the archaebacterial L12 proteins and will enable the comparison of archaebacterial and eubacterial L I2 protein folding. Together with the results from functional assays these data would complement the understanding of the function and evolution of this important protein and structure-function relationships of proteins in general.

Acknowledgments V Kruft, S Herwig, S Kielland, U Pilling, U Bergmann and D Kamp are thanked for their technical help concerning protein sequencing and amino acid analysis. The DNA sequencing of I Giinther is gratefully acknowledged. We want to thank Drs B Wittmann-Liebold and AT Matheson for their support and the improvement of the manuscript. Drs T Itoh (Hiroshima) and C Ramirez (Victoria) are thanked for their gift of the clones containing the LI2 genes of Hha and Sso, respectively. Very special thanks are due to P Leggatt for his excellent technical assistance. AKE K6pke was financially supported by a research grant from the Deutsche Forschungsgemeinschaft.

655

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