Oene. 88 (1990) 263-267 Elsevier

263

GENE 03464

Synthesis of papain in Escherichia coil (Carica papaya; protein processing; zymogen; recombinant DNA)

Larry W. Cohen, Claire Fluharty and Larry C. Dihel* 8eaver Laboratory, Deparunent of Biology, Pomona College, Claremont, CA 91711 (U.S.A.) Tel. (714)621-8000. ext. 2965 Received by G. Wilcox: 20 August 1989 Revised: 2 November 1989 Accepted: 9 November 1989

SUMMARY

We have transferred the cloned papain genetic information into an expression vector (pTT-7) regulated by the T7-promoter and have obtained in vitro expression as well as expression in Escherichia coll. In Western blots the proteins produced are immunologically recognizable as papain. Multiple forms of specific but differing sizes are detected, suggesting either that initiation can occur at more than one of the upstream methionines, or that the enzyme is processed after synthesis.

INTRODUCTION

DNA encoding the endopeptidase papain (EC 3.4.22.2) has been cloned and sequenced (Cohen et al., 1986) with the objective of engineering mutations that substitute specific aa within the structure of the enzyme. The intent is to determine the impact of the changes on substrate binding, substrate selection, catalysis and antigenicity of the protein. Papaln is excellent material for such study because it is easy to purify (Kimmel et al., 1954; Arnon, 1970; Blumberg et al., 1970; Sluyterman and Wijdenes, 1970), the x-ray crystallographic structure has been refined to 1.65!~ (Kamphuls e~ al., 1984; 1985) and a QSAR equation has Correspondence to: Dr. L.W. Cohen at his present address: California State University- San Mareos, 820 W. Los Vallecitos, San Marcos, CA 92069 (U.S.A.) Tel. (619)471-4174; Fax (619)471-4156. * Current address: Syngen, 1885 33rd Street, Boulder, CO 80301 (U.S.A.) Tel. (303)938-6200. Abbreviations: aa, amino acid(s); BMV, brome mosaic virus; MCS, multiple cloning site; nt, nucleotide(s); ORF, open reading frame; PA, polyacrylamide; PAGE, PA gel electrophoresis; Pollk, Klenow (large) fragment of E. coli DNA polymerase I; QSAR, quantitative structure activity relationship; RBS, ribosome-binding site; Rif, rifampicin. 0378-1119/90/$03.50© 1990Elsevier SciencePublishers B.V.(BiomedicalDivision)

been developed that describes the contribution of electronic, hydrophobic and bulk interactions to the binding of substrate by the enzyme (Hansch and Calef, 1976; Hansch et al., 1977; Smith et al., 1982; Carotti et al., 1984). The sequencing data had revealed that papain is probably produced as a prepro.enzyme with a 133-aa prosegment. Because the prosegment sequence contains five Met codons in ORF, it is possible that initiation occurs at various places to produce a family of zymogens with varying lengths of prosegment, hence varying characteristics. The first Met codon, however, most coincides with the eukaryotic consensus initiation sequence A/GNNATGG proposed by Kozak (1983) and only the longest sequence would contain the signal peptide portion. The aim ofthe present study was to determine whether E. coli can successfully produce the protein and whether initiation can occur at multiple sites.

EXPERIMENTAL AND DISCUSSION

(a) In vitro transcription and translation To provide regulated production of the protein, the pT7 plasmid system developed by Tabor and Richardson (1985) was employed. The vector system contains the T7 virus

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Fig. 1. Construction ofthe pT7-7 (BamHI-Pstl) and pT7-7 (HpalI-PstI) 2 0 clones. To produce the pTT-7 subclone capable of expression of the prepropapain, the MCS of the pT7-7 vector was first digested with Smal+ Pstl. To lessen the chance that the resulting small piece would reinsert in subsequent ligatiuns, the digest was heated to 85°C for 5 man and then quickly chilled. The pBR322(AT) recombinantplasmid was then cut at the unique BamHI site upstream from the prepropapain genetic information and the ends were filled-in with Pollk. The vector was cut again with PstI to release the desired piece having a blunt end and a PstI end. This piece was identified by its size on an agarose gel and purified using a modified 'freeze-squeeze' method. The purified blant-PstI fragment was finally fixated between the Sinai and Pstl sites of pT7-7. In other experiments, the fragment was cut at a unique HpalI site, filled-in with PolIk, and excised with Pail. When ligated between the Sinai and Pstl site of pT7-7, the resultant expression vector contains the information for the mature papain except that the H-terminalisoleucineis missing and replaced by information for 5 or 6 aa (depending on whether the Met remains on the completed product).

gene 10 promoter, RBS, ATG start codon and an MCS. A second plasmid in the host cells carries the phage it promoter and operator, PL and OL, adjacent to the T7 RNA polymerase gene, and is regulated by the thermosensitive c1857 repressor. Fig. 1 diagrams the construction of the expression vectors containing the genetic information for the prepro-form of papaln and of the mature enzyme. The sequence of the constructed pTT-7 (HpaII-PstI) expression vector in the vicinity of the promoter: T? promoter

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Fig. 2 shows an autoradiogram of the products of in vitro RNA polymerase transcription from the T7 promoter through the genetic information corresponding to the entire prepro-papain and subsequent translation in the presence of ~SS-labeled aa. A new band that is not present in the controls is evident at approx. 39 kDa. The size was estimated from the position of the band compared to the size standards not visible on the autoradiogram. Based on the aa sequence, as revealed by the eDNA sequence, the M r for the cloned prepropapain should be 38 945.

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Fig. 2. In vitro T7 RNA polymerase transcription and translation of genetic information corresponding to prepropapain. Modified guanine dinucleotides (GpppG) were present during transcription so that a percentage ofthe mRNAs was capped. These were then used as message in the in vitro rabbit reticulocyte translation system. Labeling by incorporation of [35S]Met and Cys was carried out for a 60-man interval at 30°C. Controls consisted of the in vitro translation system either with BMV RNA or without added RNA. The rightmost lane in the 0.1% SDS-10~ PA gel shows the pattern ofin vitro protein synthesis when using pTT-7 (BamHI.Pstl) information corresponding to prepropapain. The insert at the left ofthe figure is a shorter exposure ofthe overexposed BMV lane.

(b) in vlvo transcription end translation Fig. 3 shows an autoradiogrnm of an SDS-PA gel ofthe new products after induction of expression. The A c1857 regulation system controlling T7 RNA polymerase synthesis is 'leaky', allowing some production of the polymeruse before induction. After induction of the pT7-7 (HpalIPstl) clone, an increase is evident in bands corresponding approximately to the 24-kDa mature enzyme and an unexplained band at 29 kDa. The higher-Mr bands possibly correspond to multimers of the enzyme cross-linked by disulfide bonds. Based on the sequence of the cDNA, the exp~cted sizes of the unprocessed translation products that would result from initiation at the various prosegment Met codons, after induction of pTT-7(BamHI-Pstl) clone, would be 38945, 35742, 36702, 33 178 and 27367 Da. In the induced pTT-7(BamHI-Pstl) culture an increase of what appears to be the 39-kDa prepro-papaln was observed (Fig. 3, lanes 3 and 4). This 39-kDa size interpretation is based on the agreement between the first start codon sequence and the Kozak (1983) consensus sequence. It is possible, however, that the band represents the 38 742-Da band. Intermediate bands between 39 and 24 kDa can be

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pT7-7(BamHI-Pstl) Fi& 3. Autoradiogramof a 0.1% SDS-10%~PAgel after in vivoinduction and expression.The in vivoexpression experimentwas run in two parts. Lane !, a control induction carried out on pTT-7 (l-lpaIl.Pstl) containing cells under conditions that wouldblock the T7 polymerase from beingsynthesized.Rif(200pg/ml) had beenaddedfor 15min, then the c1857-regulatedT7 polymerasegenewas inducedfor 15min at 42°C followed by 20 rain incubation at 30°C and then a 5-rain pulse with [3SS]Met and Cys. Lane 2, results of 15-min heat induction of pTT-7 (HpalI-PstI) cells without Rif (which allowed T7 polymerase to be synthesized) followedby addition of Rif for 10min, then by 20 min of incubation at 30°Cto allowdecayof pre-existinllmRNAand, finally,by

seen which appear to be either processed forms of the prepro-enzyme or molecules synthesized beginning at the various Met codons in the prosegment. Particularly interesting is the intensity of a band at approx. 28 kDa, also observed in the experiment shown in Fig. 4A. At 35 codons upstream from the mature enzyme sequence is the sequence GATATGA (including the Met codon) with the last A being the only deviation from the Kozak (1983) consensus sequence for initiation. Initiation from that site would produce a 27 367-Da product that could be the band described above. This explanation is complicated, however, by the observation of a comparable band in the pTT-7(HpalI-Pstl) construct which lacks the upstream sequence.

(e) ImmunologicsI identificstion of the proteins as papuin The Mr of the bands strongly suggested successful in vivo production of papaln. To confn'm that the bands were papain, Western blots were carried out using antiserum prepared to papain. Fig. 4A shows a Western blot (Burnette, 1981) of uninduced and induced cultures containing the information for the prepro-enzyme (BamHI-Pstl) and the mature enzyme (HpaII-PstI) obtained in an separate experiment. Commercially marketed Mr markers, papaya fruit juice and commercial papain were run as controls. The commercial papain shows a preponderance of material at approx. 24 kDa, but a band is also visible at 39 kDa. A number of what appear to be degradation products are also present. Both 39-kDa and 24-kDa bands are also detectable in the fruit juice. In the cultures containing information for mature enzyme, a band at 24 kDa becomes evident after induction, and in the induced culture containing information for the prepro-enzyme, a band corresponding to approx. 28 kDa becomes more evident after induction. This is not the expected Mr but, as mentioned earlier, we think it corresponds either to a processed product or to a product synthesized starting at a Met codon within the prosegment other than the fu'st one. Fig. 4B shows the stained gel and the total protein complement present in the samples. The figures thus far have been of extracts of soluble protein. During bacterial production of the enzyme, however, the major amount of the papaln synthesized is apparently incorporated into insoluble intracellular particles (Fig. 5). By comparing the color densities of the bands in the Western blots to standard amounts, the yields of soluble protein are approx. 12/Lg/ml for the mature form and 2/~g/ml for the preproenzyme (Fig. 4A). Based on the densities of the bands (tenfold diluted) of the pelleted protein fraction, perhaps ten times that amount is present per liter in insoluble form. the 5.rain pulse incorporation of [3SS]Metand C'ys.Lanes 3 and 4, the corresponding experiment carried out under the same conditions on pTT-7 (BamHI.Pstl)cells.

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Fig. 5. The detection ofpapaln produced in insoluble form. Cells that had been frozen and thawed were disrupted by three passes through a French press, and the pelleted disrupted cell material was treated with lysnzyme, deoxycholate, RNase and DNase. The suspension was then spun for 5 min at S000rpm in the SV40 head. The pellets were dissolved in sample buffer and examined by 0.1Ye SDS-10% PAGE and Western blotting. DEAE and CM-Sephadex-pufified commercial enzyme was included in the gels as control to mark the papain Mr position and to identify papain in the Western blot. Fig. 4. Immunological confirmation of the in rive synthesis of papain. Uninduced and induced cultures containing the information for the prepro-enzyme and the mature enzyme were analyzed by 0.1% SDS-10% PAGE and Western blotting. (Panel A) Bands in the PA gel electroblotted to nitrocellulose, hybridized with antiserum to papain, and then hybridized with goat anti-rabbit antiserum coupled to alkaline phosphatase. Subsequent incubation with substrate (5-bromo-4-chloro-3indoyl phosphate and nitroblue tetrasolium) resulted in deposition of pigment to give the band pattern observed. (Panel B) The gel from panel A stained with Coomassie blue to show total protein.

The estimated lower yields for the prepro-form, as compared to the mature form, may be in part a reflection of the prepro-form's lower reactivity with polyclonal antiserum prepared against the mature enzyme; some of the epitopes may even be masked by the prepro-segment. In addition,

following the plasmid pT7-7(BamHI-PstI)RBS sequence and ATG, is a series of codons that contain stop codons. The next ATG corresponds to the first of the prepro-segtaunt. Once protein assembly is initiated in this clone, it probably terminates and reinitiates with lower efficiency at the papain prepro-segment Met, with the benefit of only a weak and improperly positioned RBS. The protein that is being synthesized, however, should have the correct aa sequence. Attempts to show protease activity in our recombinantproduced preparations have indicated that, with variability between experiments, activity - - if present at all - - is very low. Because the assay indicates such low background protease activity in the soluble bacterial extracts, once the papain produced is renatured, QSAR analysis on partially purified material should be feasible.

267 (d) Conclusions From the upstream sequence data it was expected that papain would be produced as a zymogen which is perhaps activated by cleavage of the N-terminal sequence. Experiments in vivo and in vitro confLrm this and also reveal that either initiation can occur at more than one site or, after synthesis, progressive processing converts the prepro-form to that found in the mature enzyme. Because of the virtual inactivity of the enzyme produced, there apparently need not be concern for the viability of the papain-producing bacterial cells, but the enzyme will have to be activated in vitro. Interestingly, it has also been reported (Brocldehurst and Kierstan, 1973) that, when produced by the plant, the enzyme may assemble with the sulfhydryl groups linked out of their active register; hence the requirement for strong reduction for it to be activated. The situation may be the same in E. coli. Many laboratories have succeeded in renaturing disuifdde cross-linked enzymes OVestfaufer et al., 1074; Pennica et al., 1983; Jaenicke and Rudolph, 1986; Marks et al., 1987; Hayakawa et al., 1987; Denefle et al., 1987; Marston, 1987).

ACKNOWLEDGEMENTS

The authors express thanks to Dr. Yechiel Becker (Hadassah Medical School, Jerusalem) for providing the Chou-Fasman analysis and to Mr. Gerhard Ott for the excellent photography. The T7 expression system was generously provided by Drs. Tabor and Richardson. This work was supported by grants from the Research Corporation and NIH (GM32687).

REFERENCES

Arnon, R.: Papain. Methods Enzymol. 19 (1970) 226-244. Binmber8, S., Schechter, I. and Burger, A.: The purification of papain by affinity chromatography. Eur. J. Biochem. 15 (1970) 97-102. Brocklehurst, K. and Kierstan, M.PJ.: Propapain and its conversion to pap,ln: a new type of zymogen activation mechanism involving intramolecular thiol-disulphide interchange. Nature New Biol. 242 (1973) 167-170. Burnette, W.N.: 'Western blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate-p01yacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Analyt. Biochem. 112 (1981) 195-~63. Carotti, A., Smith, R.N., Wong, S., l-Iansch, C Blaney, J.M. and Langridge, R.: Papain hydrolysis of X-pb~nyi-N-methanesalfonyl

81ycinatas: a quantitative structure-activity relationship and molecular graphics analysis. Arch. Biochem. Binphys. 229 (1984) ! 12-125. Cohen, L.W., Coghlan, V.C. and Dihed, LC.: Cloning and sequencing of papain-encoding eDNA. Gene 48 (1986) 219-227. Denefin, P., Kovarick, S., Gurtton, J., Cartwright, T. and Mayanx, J.: Chemical synthesis ofa gene coding for human angingenin, its expression in Esckerickia co//and conversion of the product into its active form. Gene 56 (1987) 61-70. Hansch, C. and Calef, D.F.: Structure-activity relationshipe in papain-ligand interactions. J. Or~ Chem. 47 (1976) 1240-1243. Hansch, C., Smith, R.N., Rockofl', A., Calef, D.F., Jow, P.Y.C. and Fukunaga, J.Y.: Structure-activity relationships in papaln and bromelain ligand interactions. Arch Biochcm. Biophys. 183 (1977) 383-392.

Hayakawa, T., Toibana, A., Marumoto, R., Nakahama, K., Kikuchi, M., Fujimoto, K. and Ikehura, M.: Expression ofhuman lysozyme in an insoluble form in yeast. Gene 56 (1987) 53-$9. Jaehicke, R. and Rudolph, R.: Refolding and association of oligomeric proteins. Methods EnzymoL 131 (1986) 218-250. Kamphuis, I.G., Kalk, H.K., Swarte, M.B.A. and Drenth, J.: Structure of papain refined at 1.65A resolution. J. Mol. Biol. 179 (1984) 233-256. Kamphuis, 1.(3., Drenth, J. and Baker, E.N.: Comparative study based on the high-resolution structures of papain and actinidin, and on amino acid sequence information for cathepsins B and 14, and stem bromelain. J. Mol. Biol. 182 (1985) 317-329. Kimmel, J.IL and Smith, E.L.: Crystalline papain; !. Preparation, specificity and activation. J. Biol. Chem. 207 (1954) 515-531. Kozak, M.: Comparison ofinitiation of protein synthesis in procaryntes, eocaryotes and orgenelles. MicrobioL Rev. 47 (1983) !-45. Marks, C.B., Naderi, 14., Kosen, P.A., Kuntz, I.D. and Anderson, S.: Mutants of bovine pancreatic trypsin inhibitor lacking cysteines 14 and 38 can fold properly. Science 235 (1987) 1370-1373. Murston, F.A.O.: The purification ofeukaryntic polypeptides expressed in Esckerickla coll. In Glovcr, D.M. (Ed.), DHA Cloning IlI, A Practical Approach, Chapter 4. IRL Press, Washington DC 1987, pp. 59-88. Pennica, D., Holmes, W.E., Kohr, W.J., 14arkins, R.N., Vehar, G.A., Ward, C.A., Bennett, W.F., Yelverton, E., Seeburg, P.H., 14eynekar, 14.L., Goeddel, D.V. and Collen, D.: Cloning and expression of human tissue-type plasminogen activator cDNA in E. co/L Nature 301 (1983) 214-221. Sluyterman, L. and Wijdenes, J.: An agarose mercurial column for the separation of mercaptopapaln and non-mercaptopapaln. Biochim. Biophys. Acta 200 (1970) 593-595. Smith, R.N., l-lansch, C., Kim, K.H., Omiya, B., Fukumura, G., Selassie, C.D., Jow, P.Y.C., Blaney, J.M. and Lanwidge, R.: The use ofcrystalIography, graphics and quantitative structure-activity relationships in the analysis of the papain hydrolysis of X-phenyl hippurates. Arch. Binchem. Biophys. 215 (1982) 319-328. Tabor, S. and Richardson, C.C: A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82 (1985) 1074-1078. Westfaufer, D.B., Johnson, E.R. and Clanss, L.M.: Rapid non-enzymatic regeneration of reduced human leukemia lysozyme. In Osserman, E.F., Canford, R.E. and Beychok, S. (Eds.), Lysozyme. Academic Press, New York, 1974, pp. 269-280.