Protein Engineering vol.3 no.7 pp.635-639, 1990

Expression, purification and crystallization of penicillin G acylase from Escherichia coli ATCC 11105

P.D.Hunt1'2, S.P.ToUey, R.J.Ward, C.P.Hffl3 and G.G.Dodson Department of Chemistry, University of York, Heslington, York YO1 5DD, UK 'Present address: Department of Biology, University of York, Heslington, York YO1 5DD, UK 'Present address: Molecular Biology Institute, UCLA, Los Angeles, CA 90024, USA 2

To whom correspondence should be addressed

Introduction Penicillin G acylase (PA, benzylpenicillin acylase, penicillin amidase, penamidase, acyl transferase, penicillin splitting and synthesizing enzyme, benzylpenicillin amidohydrolase; EC 3.5.1.11) catalyses the hydrolysis of penicillin G (benzyl penicillin) to 6-aminopenicillanic acid (6-APA) and phenylacetic acid. As 6-APA is the basis for the synthesis of many novel penicillins the enzyme is of industrial and commercial interest. The active form of the enzyme is found in the periplasm of Escherichia coli (Mayer et al., 1979). It consists of two subunits of mol. wt 24 000 (a) and 65 000 03), produced by proteolytic cleavage of a precursor molecule to remove an N-terminal signal peptide and a 54-amino-acid spacer peptide linking the two subunits (Schumacher etai, 1986). The requirement for processing has made overproduction of the enzyme for crystallographic studies difficult. The gene (pac) encoding PA from E. coli ATCC 11105 has been cloned into the vector pBR 322 and its derivatives (Mayer et al., 1979; Oliver et al., 1985). Penicillin acylase genes from E.coli 194, Bacillus megaterium (Meevootisson and Saunders, 1987), Arthrobacter viscosus (Ohashi et al., 1988), and Kluyvera citrophila (Garica and Buesa, 1986) have been cloned into pACYC 184. However, cells containing these constructs do not produce large quantities of the enzyme. Schumacher et al. (1986) describe the construction of a plasmid which encodes the E. coli ATCC 11105 PA precursor without a signal sequence, under the control of the tac promoter. On induction, strains carrying this plasmid accumulate the PA precursor in soluble form in the cytoplasm. When the cells are lysed the precursor is processed, presumably by a periplasmic enzyme which only comes into contact with the precursor after

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Materials and methods Materials 6-Nitro-3-phenylacetamidobenzoic acid (NIPAB), polymyxin B sulphate, polyethylene glycol 8000, trizma base, 3-(Nmorpholino) propanesulphonic acid (MOPS), benzamidine hydrochloride, phenylrnethylsulphonylfluoride (PMSF), deoxyribonuclease I (DNase I) and lysozyme were obtained from Sigma Chemical Company. 5-Bromo-4-chloro-3-indolyl-/3-D-galactoside (X-gal) and isoproylthio-/3-galactoside (IPTG) were purchased from Gibco BRL. Goat anti-rabbit horseradish peroxidase conjugated antiserum was purchased from Bio-Rad Laboratories Ltd. Rabbit anti-penicillin acylase antiserum was the generous gift of D.Sizmann (Lehrstuhl fur Mikrobiologie der Universitat Munchen, Munchen, FRG). Partially purified penicillin G acylase was the gift of Dr G.Schumacher, Boehringer Mannheim GmbH, Tutzing, FRG. All other chemicals were of analytical reagent grade and purchased from Fisons PLC, UK. Bacterial strains and plasmids Escherichia coli JM 109 is (rec Al, end Al, gyr A96, thi, hsdR17 r K - mK + , sup E44, rel Al, A(lac-pro AB) (F'tra D36, pro AB, lac IqZ A M15)) (Hanahan, 1985); E.coli 5 K (rK- mK + , thr, thi) was supplied by Dr J.Collins (GBF, Braunschweig, FRG) and E.coli G6 (Hfr his thi) was the gift of Dr S.J.S.Hardy (Department of Biology, University of York). Plasmid pUC 9 (Viera and Messing, 1985) was obtained from Gibco BRL Ltd, UK. pPLc2833 (Remaut et al., 1981) and pC1857 were supplied by Dr Erik Remaut (Laboratory of Molecular Biology, State University of Ghent, Belgium). pHM12 (Mayer et al., 1979) was the gift of Dr J.Collins, and is a Pstl-EcoRl fragment containing the pac gene from the chromosomal DNA of E. coli ATCC 11105 cloned into the vector pBR 322 (Figure 1). Methods Manipulation of DNA. Restriction digests, agarose gel electrophoresis, ligation, extraction and purification of plasmid DNA were all performed essentially as described by Maniatis etal. (1982). 635

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The penicillin acylase gene (pac) from Escherichia coli ATCC 11105 was cloned into pUC 9 and the resulting vector (pUPA-9), when transformed into E.coli strain 5K, allowed the constitutive overproduction of mature penicillin acylase when grown at 28CC. The enzyme ws purified from the periplasmic fraction of E.coli pUPA-9 by hydrophobic interaction chromatography and anion exchange. Crystals of penicillin acylase were grown in batch using polyethylene glycol 8000 as a precipitant. The crystals (space group PI) diffracted to beyond 2.3 A. Key words: penicillin acylase/protein crystallization/overproduction of protein

lysis. Unfortunately, no data are given as to the yield of PA obtained in this system. In order to grow crystals for X-ray analysis of the enzyme, quantities of the order of 10-50 mg of pure protein were necessary. Originally we purified the enzyme from a sample of E.coli ATCC 11105 lysate supplied by Glaxo PLC, UK. Crystals grown from penicillin acylase purified from this source were of low quality. Partially purified penicillin acylase was also supplied by Boehringer Mannheim GmbH. This was further purified using the methods described here and produced good crystals. In order to have a reliable source of penicillin acylase for crystallization and to express mutated forms of the enzyme a highlevel expression system was required. In this paper we describe the development of such a system and the subsequent purification and crystallization of the enzyme.

P.D.Hunt et al.

Immunoblotting. Immunoblotting was carried out according to the procedure described by Davis et al. (1986), probing with a rabbit anti-penicillin acylase polyclonal antiserum, and a goat antirabbit horseradish peroxidase conjugated antiserum. Cloning of penicillin acylase gene. Plasmid PAPPL was constructed by cloning a 3.4-kb HindTQ fragment containing the PA genes from plasmid pHM 12 into the expression vector pPLc 2833 (Remaut et al., 1981). Ligated plasmids were transformed into stain G6 pC1857 and transformants were selected for Hind III

ampicillin and kanamycin resistance. Plasmids were prepared from transformants and screened for the presence of inserts in the correct orientation by digestion with EcoRl. A map of pAPPL is shown in Figure 1. Plasmid pUPA-9 was constructed by cloning a 6.2 kb EcoRl—Pstl fragment from plasmid pHM 12 into the vector pUC 9 previously digested with PsA and EcoRl. Ligated plasmids were transformed into E.coli strain JM 109 according to the method of Hanahan et al. (1985). Transformants were selected on LB plates containing 0.005% X-gal, 1 mM IPTG and 0.2 mg/ml ampicillin. Those transformants forming white colonies were screened for penicillin acylase activity by growing overnight at 30°C in 2 X TY medium containing 200 /ig/ml ampicillin, then spheroplasting the cells. Periplasmic fractions so obtained were assayed for penicillin acylase activity. In those isolates which showed penicillin acylase activity the spheroplasts were used to prepare plasmid DNA. This produced a plasmid of — 8.9 kb as expected. The plasmid was digested with various restriction enzymes to confirm the restriction map shown (Figure 1). Results Expression of penicillin acylase Cultures of E.coli G6 pC1857 pAPPL were grown in 2 X TY medium containing 200 /ig/ml ampicillin and 50 /ig/ml 1

2

3

4

5

6

7

B

9

10

Hpal

it**

TetR

Hpal

EcoRl Hind III

Fig. 2. Induction of synthesis of PA precursor in E.coli G6 pC1857 pAPPL. Cells were grown and induced as described in the text. Lane 1, mol. wt markers 97 400, 66 000, 45 000, 36 000, 20 000 and 14 000; lane 2, uninduced cells; lane 3, cells 30 min after induction; lane 4, cells 60 min after induction; lane 5, cells 90 min after induction; lane 6, cells 120 min after induction; lane 7, cells 150 min after induction; lane 8, cells 180 min after induction; lane 9, purified PA; lane 10, mol. wt markers as in lane 1. Equal numbers of cells were loaded in lanes 2 - 8 .

AmpR

Table I. Strain dependence of expression of penicillin acylase

Fig. 1. Expression plasmids used in this study: pHM12 was the gift of Dr J.CoUins (GBF, Braunshwcig, FRG), and is a Pstl-EcoRl fragment from the genomic DNA of E.coti ATCC 11105 cloned into pBR322. Plasmid pUPA-9 was constructed by cloning this Pstl-EcoRl fragment from pHM 12 into pUC 9. In pAPPL the pac gene is under the control of the XPL promoter. This was achieved by cloning a 3.4 kb HindSH fragment into the multiple cloning site of the vector pPLc 2833. Plasmids are not drawn to scale and only relevant restriction sites are shown.

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Strain

Penicillin acylase (U/108 cells)

5K pHM 12 JM 109 pUPA-9 5K pUPA-9

0.0138 0.0086 0.0940

Penicillin acylase activity was determined as described in the text. All cultures were grown at 28°C in 2 x TY medium containing 200 mg/1 ampkillin.

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Extraction of periplasmic proteins. Cells were harvested by centrifugation and resuspended in ice-cold 0.3 M sucrose/0.1 M Tris—HC1, pH 8.5/0.1 mg/ml polymyxin B sulphate. Lysozyme was added to a final concentration of 0.2 mg/ml and the cells incubated on ice for 30 min. After centrifugation at 20 000 g for 10 min at 4°C, the supernatant containing the periplasmic proteins was collected. Lysis of cells. Cells were lysed by suspension in 50 mM Tris-HCl, pH 7.8, adding Mg 2 * to 5 mM, PMSF to 0.25 mM, benzamidine hydrochloride to 25 nM and DNase I to 25 /ig/ml. The suspension was sonicated for 60 s on ice using an MSE ultrasonic disintegrator. SDS-PAGE. SDS-PAGE gels were run according to the method of Laemmli (1970). Assay of penicillin acylase activity. Penicillin acylase activity was assayed by a modification of the method of Kutzbach and Rauenbusch (1974). Samples were added to a solution of 0.15 mM 6-nitro-3-phenylacetamidobenzoic acid (NIPAB) in 50 mM sodium phosphate buffer, pH 7.5, and the change in absorbance at 410 nm measured. One unit of enzyme activity is defined as the quantity catalysing the hydrolysis of 1 /nmol of substrate per min at 25 °C.

Penicillin G acyiase from E.coli ATCC 11105

%FidScml< PAactMty

of both periplasms were analysed by SDS—PAGE, which revealed that apparently identical release of periplasm had occurred in both strains. To determine if this behaviour was an effect of the different host strains plasmid pUPA-9 was transformed into strain 5K. The resulting strain 5K pUPA-9 was grown overnight at 28°C and spheroplasted as described above. The periplasm was assayed for penicillin acylase activity, and the results are shown in Table I. It can be seen that the activity of PA in the periplasm of 5K pUPA-9 is considerably increased compared with that of either JM 109 pUPA-9 or 5K pHM12, demonstrating the importance of the host strain in expression or export of penicillin acylase. Expression of PA in strain 5K pUPA-9 was also found to be temperature dependent. Maximum levels of expression were found at 28 °C, with PA production being completely repressed at 40°C. The mechanism of temperature dependence is not known; however, periplasmic extracts of cells grown at higher temperatures contain PA precursor, as demonstrated by immunoblotting (data not shown), so it is possible that processing is inhibited at higher temperatures. Purification of penicillin acylase Ten litres of 2 x TY containing 0.4 g/1 ampicillin were inoculated with 5K pUPA-9 and shaken at 28°C for 20 h. The cells were harvested by centrifugation, and spheroplasted in a final volume of 1 1. Spheroplasts were removed by centrifugation. Subsequent procedures were carried out at room temperature. Ammonium sulphate was added to the periplasm to a final concentration of 1.7 M. Precipitated proteins were removed by filtration, and the filtrate loaded onto a 114 ml fast flow phenyl

6

PAaoMty

Fig. 3. Upper. Purification of penicillin acylase from 5K pUPA-9 periplasmic extract by chromatography on fast-flow phenyl Sepharose, as described in the text. Lower. Purification of penicillin acylase on FPLC Mono-Q column, as described in the text. In each case 100% full = ^280 ™n °f ' u - Penicillin acylase was assayed as described.

Fig. 4. 0.1% SDS/12% polyacrylamide gel showing the purification of penicillin acylase from E.coli 5K pUPA-9. Lanes 1 and 6, mol. wt markers 67 000, 45 000, 36 000, 29 000, 24 000, 20 000 and 14 000; lane 2, periplasmic extract; lane 3, pooled active fractions from phenyl Sepharose column; tone 4, pooled peak fractions from FPLC MoncKJ column; lane 5, purified penicillin acylase supplied by Boehringer Mannheim. In lanes 2—5 0.02 U of PA activity were loaded per lane.

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kanamycin at 30°C to an A5(a „„, of 1.0. The temperature was then raised to 42 CC and the cells grown for a further 2 h. Cells were harvested by centrifugation and lysed by sonication as described above. The cell lysate was analysed by SDS-PAGE and assayed for penicillin acylase. No penicillin acylase activity was detectable. Gels showed induction of a major band of mol. wt ~ 90 kb (Figure 2). This band cross-reacts with anti-penicillin acylase antiserum and can therefore be identified as the penicillin acylase precursor (Schumacher et al., 1986). The protein was found in the pellet on centrifugation at 12 000 g for 5 min, but could be solubilized by 8 M urea, indicating that it was forming inclusion bodies. Also, when induced cells were examined by phase-contrast microscopy highly refractile bodies were seen, which were not present in uninduced cells. This also indicated the presence of inclusion bodies (data not shown). Overnight cultures of strains JM 109 pUPA-9 and 5K pHM12 were grown at 30°C in 2 X TY containing 200 /ig/ml ampicillin or 20 /ig/ml tetracycline respectively. Periplasmic fractions were assayed for penicillin acylase activity. Results are shown in Table I. It can be seen that JM 109 pUPA-9 produces less penicillin acylase than 5K pHM12. As pUC 9 has a much higher copy number than pBR 322, on which pHM 12 is based, this was unexpected. To check that the lower yield of PA was not the result of variation in the efficiency of spheroplasting, samples

P.D.Hunt et al.

Sepharose column (Pharmacia), which was then washed with 200 ml of 1.7 M ammonium sulphate/50 mM Tris-HCl, pH 7.0. No penicillin acylase activity was detectable in the column effluent. Bound material was then eluted using a gradient of 1.7-0 M ammonium sulphate in 50 mM Tris-HCl, pH 7.0, over a volume of 150 ml, followed by washing with 200 ml of 50 mM Tris—HC1, pH 7.0. Fractions were assayed for penicillin acylase activity, which was found to elute in the first 30 ml after

Table D. Purification of penicillin acylase Stage of purification

Sp. act. (U/mg)

% Recovery

Periplasmic extract FFPS peak Mono Q peak

0.2 6.1 15.38

100 88 72.3

Fig. 5. (A) Penicillin acylase crystals grown as described in the text. (B) 6° precession photograph of a penicillin acylase crystal.

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Penicillin acylase activity and protein concentration were determined as described in the text. FFPS = fast flow phenyl Sepharose.

the end of the gradient. Fractions containing penicillin acylase were pooled and dialysed for 16 h against 2 1 of 50 mM Tris—HC1, pH 8.5. The contents of the dialysis bag were centrifuged at 20 000 g for 10 min to remove precipitated material. The supernatant was loaded onto a Pharmacia HR 10/10 Mono-Q column previously equilibrated with 50 mM Tris-HCl, pH 8.5, and eluted with a gradient of 0 - 7 5 mM NaCl in 50 mM Tris-HCl, over a volume of 200 ml. Penicillin acylase activity eluted as a single peak between 45 and 60 mM NaCl (Figure 3). After the end of the gradient the column was washed with 25 ml of 0.5 M NaCl/50 mM Tris-HCl, pH 8.5. Another peak eluted but contained no PA activity. The fractions from the peak containing PA activity were analysed by SDS—PAGE, and found to contain two bands of mol. wts corresponding to those of the two subunits of penicillin acylase (Figure 4). These fractions were pooled, and the protein concentration determined. They were assayed for penicillin acylase activity and the sp. act. calculated to be 15.38 U/mg protein. Electrophoretically pure PA derived from material supplied by Boehringer Mannheim GmbH was found to have a sp. act. of 13.27 U/mg.

Penicillin G acylase from E.coli ATCC 11105

Preliminary crystallographic analysis Crystals were investigated initially using screened precession photographs, which showed the space group to be PI. Cell dimensions were determined a& a = 5X.1 K, b = 64.7 A, c = 75.9 A, a = 100.6°, 0 = 111.2°, y = 105.8°. Assuming that there is one heterodimer of 89 kd per asymmetric unit, the specific volume (VM) is 1.5 A3/dalton, corresponding to a solvent content of - 50% (Matthews, 1968). The crystals diffract to beyond 2.3 A . A precession photograph is shown in Figure 5. Discussion We describe here the overproduction of penicillin acylase in E.coli by constitutive synthesis of the protein from the pac gene cloned into a high copy number vector. In this system the expression of penicillin acylase is controlled by the promoter of the pac gene, as no extrinsic promoters are present. The high gene copy number resulting from the use of pUC 9 rather than pBR 322 (or its derivatives) allows the production of penicillin acylase at levels in excess of 10 mg/1 of cells, a 7-fold increase in expression over 5K pHM 12. This latter strain itself produces 40-fold more PA than E.coli ATCC 11105 (Mayer et al., 1979). In this system the penicillin acylase is exported to the periplasm of the cell and is in the mature, active form. This has allowed us to circumvent the problem of processing overproduced penicillin acylase precursor, enabling us to purify the enzyme from periplasmic extracts. As >70% of the total PA can be recovered in pure form, the final yield of protein per litre of culture compares favourably with that obtained for other proteins expressed from strong inducible promoters. In the latter systems the necessity for solubilizing inclusion bodies and subsequent refolding of the protein often leads to substantial losses (Dodson et al., 1988; Marston et al., 1984). Constitutive expression of PA leads to the accumulation of the processed form of the enzyme, presumably by avoiding saturation of the processing pathway. By contrast, the use of a XPL-based high-expression system led to the accumulation of inclusion aggregates of PA precursor complete with the signal sequence, indicating that export of the protein was prevented. The constitutive expression system described might provide a good

expression system for other exported proteins which also require cleavage of the signal sequence, though it would obviously be unsuitable for proteins which are toxic to E.coli. The use of this expression system has permitted purification of sufficient enzyme for the growth of crystals, which are being used to determine the structure of the enzyme by X-ray diffraction methods. This in turn will allow the design of mutated proteins with altered properties, e.g. temperature stability, pH optimum and substrate specificity, all of which could increase the enzyme's industrial usefulness. The constitutive expression system described here will also facilitate experiments on various aspects of the molecular biology of penicillin acylase, e.g. further study of the processing pathway of the enzyme. The proteolytic processing of the precursor to produce the mature enzyme occurs in a way which is apparently unique in prokaryotes (Schumacher et al., 1986), although there are parallels with eukaryotic proteins, e.g. insulin. According to Schumacher et al. (1986) the /3 subunit is first cleaved from the precursor, leaving the connecting peptide attached to the a subunit. The connecting peptide is then removed in two distinct stages to produce the mature a subunit. It is not known wnether this process occurs after export, i.e. in the periplasm, or simultaneously with the export process There is some evidence to suggest that the latter is the case (Bock et al., 1983). The expression system we describe here should allow us to elucidate further details of this pathway. Acknowledgements The authors would like to thank Glaxo PLC, UK, and Dr Gunter Schumacher of Boehringer Mannheim GmbH, FRG for initial supplies of penicillin acylase, Steven Smerdon for preliminary experiments, Dr Douglas R.DTummond for the supply of bacterial strains and Dr Peter Moody, Helen Swift and Dr S.J.S.Hardy for helpful discussion and assistance with the preparation of this manuscript. This work was supported by a grant from the SERC Protein Engineering Initiative.

References

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The purification is summarized in Table n, showing yields and increasing specific activities in successive stages. Crystallization of penicillin acylase Crystals of penicillin acylase were obtained from a 12 mg/ml solution of the protein in 50 mM MOPS buffer, pH 7.2, using batch techniques, other techniques (hanging drops, dialysis or microdialysis) having been unsuccessful. Solid polyethylene glycol 8000 (PEG 8000) was added to the protein solution with continual stirring until a slight precipitate was formed (10-12% w/v PEG). The precipitate was then redissolved by the addition of the small aliquots (20-50 /d) of 50 mM MOPS, pH 7.2. The solution was filtered through 0.2 /itn filters into small glass tubes, sealed and allowed to crystallize at 20 c C. Crystals usually appeared within 2 - 3 weeks. Once crystals had been obtained the crystallization was made more reliable by employing seeding techniques, adding one or two washed small crystals to protein/PEG solutions made as described above. Using this method single crystals suitable for X-ray analysis were obtained, usually within 3—4 days. Average crystal dimensions were 0.4 x 0.3 X 0.2 mm. Occasionally very large crystals, e.g. 7 x 2 x 1 mm were obtained but these were generally twinned and not of adequate quality for X-ray analysis. A photograph of a typical crystal is shown in Figure 5.

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