disulphide linkages are a structural component of ... - Europe PMC

The EMBO Journal vol.4 no.3 pp.775-780, 1985

Examination of calf prochymosin accumulation in Escherichia coli: disulphide linkages are a structural component of prochymosincontaining inclusion bodies

J.M.Schoemaker1, A.H.Brasnett2 and F.A.O.Marston3 Department of Molecular Genetics, and 3Department of Protein Biochemistry, Celltech Ltd., 250 Bath Road, Slough SLI 4DY, UK 'Present address: Department of Biochemistry and Molecular Biology, The University of Chicago, Cummings Life Science Center, 920 East 58th Street, Chicago, IL 60637, USA 2Present address: Department of Genetics and Biometry, University College London, 4 Stephenson Way, London, UK

Communicated by D.C.Phillips

Recent reports have shown that synthesis of certain recombinant proteins in Escherichia coli results in the production of intracellular inclusion bodies. These studies have not analyzed the structure of the inclusion body especially regarding the intermolecular forces holding it together. We have examined structural aspects of inclusion bodies made in E. coli as a result of high level expression of the eukaryotic protein, calf prochymosin. Prochymosin is a monomeric protein containing three disulfide bridges. It was expressed at up to 20% of cell protein from a plasmid containing the E. colU tryptophan promoter, operator and ribosome binding site. Proteins in the inclusion bodies were analysed by Western blotting of SDS-polyacrylamide gels. When experiments were done using conditions which preserved the in vitro state of thiol groups, inclusions were shown to be composed of multimers of prochymosin molecules which were interlinked partly by disulfide bonds. The inclusion bodies also contained a high concentration of reduced prochymosin. The presence of intermolecular disulfides probably contributes to the difficulty of solubilizing recombinant prochymosin during its purification from E. coil. Key words: disulfides/inclusion bodies/industrial enzyme/prochymosin/recombinant protein Introduction Since the advent of recombinant DNA technology a variety of eukaryotic and procaryotic genes have been cloned and expressed in bacteria (Harris, 1983). However, we lack basic knowledge of how the bacterial cell copes with large concentrations of foreign proteins in its cytoplasm. We have examined the effects of high level expression of the eukaryotic protein, prochymosin in E. coli. Prochymosin is the zymogen of the milk clotting enzyme chymosin (rennin), which is commercially important in the process of cheese-making. Recently, prochymosin cDNA has been cloned in a plasmid containing the E. coli tryptophan promoter, operator and ribosome binding site (Harris et al., 1982; Emtage et al., 1983; Nishimori et al., 1984). We show that prochymosin is expressed at high levels in E. coli strain B/r and, concomitant with this, intracellular inclusion bodies appear. These have been isolated intact from the cell and shown to be composed of aggregates of prochymosin molecules which are interlinked at least partly by disulfide bonds. The inclusion bodies also contain a high concentration of fully reduced prochymosin. IRL Press Limited, Oxford, England.

Results Morphological effects of high level expression of prochymosin in E. coli B/r E. coli strain B/r was transformed with plasmid, pCT70, which encodes the prochymosin gene downstream from the E. coli tryptophan promoter, operator and ribosome binding site (Harris et al., 1982). Strain B/r was used because it accumulated more prochymosin than the E. coli K-12 strains tested (unpublished results). Maximal accumulation of prochymosin was 20% of total cell protein. Throughout the growth of a culture in glycerol induction medium, cell samples were periodically inspected by phase contrast microscopy. In early exponential phase small dark inclusion bodies appeared in the cytoplasm. These increased in volume and became highly refractile by mid-exponential phase (Figure 1). Refractile inclusions were not detectable under the same conditions in E. coli B/r cells containing the parent plasmid, pCT54, which encodes the tryptophan control regions but no prochymosin DNA sequences. The inclusions generally localised at the polar or sub-polar regions, with a large percentage of normal-length cells having one incluson near each pole. Some other types of inclusions which are found in bacteria, such as glycogen accumulations, also tend to localise at the polar regions (Schoemaker et al., 1981; Shively, 1974). This may simply result from the fact that large accumulations are displaced to areas of the cytoplasm which are not occupied by other major structural entities such as the chromosome. Electron microscopic investigations of thin sections of cells containing inclusions, stained with common electron microscopic stains, revealed dense amorphous bodies which did not appear to be surrounded by membranes but tended to have discrete boundaries with the rest of the cytoplasm (Figure 2). Lack of birefringence of the inclusion bodies indicated that they are not crystalline. Analysis ofprochymosin in whole cell extracts by Westem blotting To examine the in vivo state of prochymosin accumulation, especially with regard to disulfide bond formation, cell extracts of E. coli B/r cells showing large intracytoplasmic inclusion bodies were analysed on Western blots using polyclonal rabbit anti-prochymosin antibody. Observations were also made on the control, E. coli B/r containing the parent plasmid pCT54 (lacking prochymosin DNA). To preserve the in vivo state of thiol groups, cell samples (determined by optical density) were lysed by boiling for 5 min in degassed Tris-SDS buffer containing 35 mM iodoacetamide in sealed tubes under N2. Iodoacetamide will alkylate free thiols preventing further oxidation of these residues. Reaction mixtures were incubated for 50 min at room temperature and then equal concentrations were loaded on gels immediately. Figure 3 is a Western blot (see Materials and methods) of these cell preparations. Electrophoresis was carried out in the presence (lanes 8- 14) or absence (lanes 1 -7) of the thiol reducing agent, 2-mercaptoethanol. A comparison of the oxidised and reduced forms of pure -

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recombinant prochymosin is shown in lanes 5 and 12, respectively. In the oxidized state, when intramolecular disulfides are formed [prochymosin has three disulfide linkages (Foltmann et al., 1977)], the molecule presumably has a more compact structure leading to a noticeably faster mobility. The slightly larger band of the doublet which appears in lane 5 (and possibly 6), which also runs faster than the reduced prochymosin band in lane 12, probably represents incompletely oxidised prochymosin molecules. The antibody is shown to be specific for prochymosin in lanes 5-7 and 12-14. Purified recombinant prochymosin was added to cell extracts of E. coli B/r containing the parent plasmid, pCT54 in a Tris-SDS lysis buffer in the absence (lanes 5 and 12) or presence (lanes 6 and 13) of iodoacetamide. No prochymosin-sepcific bands were detectable either above or below the pure prochymosin band and no bands were visible at all in the control lanes 7 and 14 containing only E. coli B/r/pCT54. When cells extracts were run on polyacrylamide gels under non-reducing conditions (lanes 1-4), low mol. wt. prochymosinspecific bands ( < 40 000), as well as a large size range of high mol. wt. (>40 000) prochymosin aggregates were detectable, including a very large aggregate (> 100 000) which did not enter the stacking gel (uppermost band in lanes 1-4). The low mol. 776

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Fig. 3. Western blot of lysate of cells containing either pCT54 (control) or pCT70. Lanes 1-7, no 2-mercaptoethanol in electrophoresis sample buffer; lanes 8-14, with 2-mercaptoethanol. Lanes 1 and 8, pCT70-containing cells lysed under N2 in Tris-SDS buffer with iodoacetamide. Lanes 2 and 9, pCT70-containing cells lysed under N2 in same buffer lacking iodoacetamide. Lanes 3 and 10, pCT70-containing cells lysed in air in same buffer with iodoacetamide. Lanes 4 and 11, pCT70-containing cells lysed in air in same buffer lacking iodoacetamide. Lanes 5 and 12, pCT54-containing cells lysed in same buffer lacking iodoacetamide but with added pure prochymosin. Lanes 6 and 13, pCT54-containing cells lysed in same buffer with iodoacetamide and added pure prochymosin. Lanes 7 and 14, pCT54-containing cells lysed in same buffer lacking idoacetamide and no added pure prochymosin. All pCT54-containing cells were lysed in air. re = reduced prochymosin, ox = oxidised prochymosin. wt. bands below the prochymosin monomer band (i.e.,

40 000) aggregates concomitant with a may represent

greater intensity of the reduced prochymosin band (Figure 3, lanes 8-11). The intensities of the low mol. wt. bands (40 000 mol. wt.) to reduced prochymosin (arrow) in lane 1, lysate of pCT70-containing cells with large inclusions, and lanes 2 and 3 increasing concentrations of a lysate of pCT66-containing cells, with no visible inclusions. Lane 4 is a control on antibody specificity and represents a lysate of pCT54-containing cells.

chymosin band (arrow). Very little prochymosin was visible in the supernatant fraction, resulting from the separation of inclusion bodies from the remainder of the cell lysate by centrifugation (lanes 4-5), even when the supernatant fraction was loaded at twice the relative volume of the inclusion fraction (compare lanes 3 and 5).

Disulfide

Examination ofprochymosin in cells with no visible inclusion bodies We examined whether disulfide-linked multimers of prochymosin could be detected in cells with no visible inclusion bodies. Figure 6 is a Western blot of strain B/r containing the plasmid pCT66 which has a different distance between the Shine-Dalgarno sequence and the ATG of the ribosome binding site from that in pCT70, resulting in decreased translation (Emtage et al., 1983). No intracellular inclusions were visible with phase contrast microscopy at any time during the growth cycle. Cell extracts prepared in the standard Tris-SDS buffer containing iodoacetamide and electrophoresed under non-reducing conditions were compared with those from B/r/pCT70 which had large inclusions. In B/r/pCT66 (lanes 2 and 3) very little aggregated (disulfide-linked) prochymosin was detectable relative to reduced prochymosin, in contrast to B/r/pCT70 (lane 1) in which the relative amount of aggregated polypeptide to reduced polypeptide is much higher. The only visible aggregated protein in lane 3 appears to have the mol. wt. of dimers (-80 000).

Discussion Several reports have emerged showing that certain other recombinant proteins, which accumulate to high levels in E. coli, produce intracellular inclusion bodies (Klier et al., 1982; Weis et al., 1983; Williams et al., 1982; Wetzel and Goeddel, 1982). However, these studies have not analyzed the structure of the inclusions in vivo with regard to the presence of disulfide linkages. We have examined the composition of inclusion bodies which result from a high level of accumulation of the eukaryotic protein, prochymosin, in E. coli B/r. On Western blots, inclusions were shown to be composed of a large size range of prochymosinspecific polypeptides including variously sized aggregates of polypeptides (mol. wt. >40 000) as well as a large concentration of fully reduced prochymosin. Disulfide linkages were present in the aggregates as indicated by their disaggregation on heating in the presence of 2-mercaptoethanol. Prochymosin is a single peptide chain containing six halfcysteine residues. Two or more molecules could interact to form disulfide linkages between one or more of these cysteines thus giving dimers, trimers, etc., resulting in some of the high mol. wt. bands (> 80 000) visible on Western blots. In addition, the low mol. wt. (40 000 on Western blots. Heating cell extracts containing inclusion bodies in a Tris-SDS buffer alone also caused some disaggregation of prochymosin aggregates (Figure 4). This suggested that other non-covalent types of intermolecular forces were involved in holding the molecules together, including binding the fully reduced prochymosin to the inclusion body. Washing inclusion bodies in a buffer containing Trixon X-100 did not appreciably solubilize prochymosin, suggesting that hydrophobic bonds alone are probably not responsible for holding the reduced molecules to the inclusion body (Marston et al., 1984). Cells which made only a small amount of prochymosin and had no visible inclusion bodies (E. coli B/r/pCT66) showed a higher concentration of reduced prochymosin relative to aggregated (disulfide-linked) molecules, than cells containing large inclusion bodies (E. coli B/r/pCT70). The observation of mainly reduced prochymosin in pCT66-containing cells is consistent with the fact that, in general, the cytoplasm of E. coli is reduc-

linkages in prochymosin-containing inclusions

ing and most intracellular proteins are rich in thiols (Freedman and Hillson, 1980). However, the question arises as to how disulfides are formed in pCT70-containing cells. One possible explanation is that the large concentration of prochymosin, represented by the inclusion body, may simply be in excess of the available reducing equivalents. Another possiblity is that the inclusion body may be a microenvironment which facilitates the formation of disulfide linkages (both intra and intermolecularly) because (i) the molecules within the body would be sequestered from the reductive cytoplasm and (ii) they would be densely packed and, therefore, more likely to interact. Clearly, further experiments are necessary to examine these possibilities or others which could explain the formation of disulfide linkages in vivo, and to determine the importance of these linkages in the formation of the inclusion body. Almost no low mol. wt. polypeptides (below the reduced prochymosin band) were visible in cells harboring pCT66, as compared with pCT70-containing cells (Figure 8). This may be because translation from pCT66 is reduced as compared with pCT70 and, therefore, the tendency to form abnormal translation products may be less with the former plasmid. Alternatively, if some of the low mol. wt. polypeptides are intermediate degradation products they may be protected from further degradation by being sequestered in the large inclusion body in pCT70-containing cells. The presence of disulfide linkages in the inclusion body may be partially responsible for the insolubilty of recombinant prochymosin, i.e., it cannot be readily solubilized by the common agents that would disaggregate molecules joined by weaker molecular forces (Marston et al., 1984). Certain other recombinant proteins, which produce intracellular inclusion bodies, have been shown to be associated with the insoluble fraction of E. coli, i.e., they are found in the pellet resulting from cell lysis and centrifugation (Harris, 1983). It is likely that disulfides are structural components of the inclusions associated with some of these proteins, and, therefore, may also contribute to the difficulty in solubilizing these recombinant proteins when purifyng them from the E. coli cell. Materials and methods Media and growth conditions Cells were grown overnight in Luria broth with 100 /g carbenicilin/mi. A 1:100 dilution of the overnight culture was made into glycerol induction medium lacking tryptophan [for induction of the tryptophan operon (Yanofsky, 1981)] containing per litre: 3 g KH2PO4, 6.0 g Na2HP04, 0.8 g NH,4Cl, 0.1 g NaCl, 0.004 g CaCl2.6H20, 0.05 g MgSO4.7H20, 3.2 g glycerol, 0.2 g yeast extract, 3 g Vitamin Assay Casamino Acids (Difco Laboratories), 10 mg thiamine, and 0.1 g carbenicillin. Light and electron microscopy Suspension cultures were analysed on 1 mm thick M9 agar blocks (10 mm x 10 mm) (Schoemaker et al., 1981), and were photographed using a Nikon Labophot microscope equipped with phase contrast optics. Kodak Plus X PAN film was used for photography. Standard fixation, embedding and staining techniques for electron microscopy were carried out as in (ibid). Thin sectioning and photography was done on a JEOL CX 200 electron microscope in collaboration with Naomi Nicholas, Department of Crystallography, Birbeck College, University of London, Malet Street, London, UK. Western blotting Western blotting was carried out as described (Towbin et al., 1979) and subsequently modified (Burnette, 1981). The nitrocellulose blot was probed with rabbit polyclonal anti-prochymosin antibodies and ['251]Protein A. Antibodies were prepared by Julia Spragg at Celltech Ltd. Cell extracts for Western blotting were prepaed as follows. To preserve the in vivo state of thiol groups, cells were lysed by heating at 95°C for 2-5 mmn in a buffer containing 100 mM Tris Cl pH 6.5, 5 mM EDTA, 15 mg/mi SDS, 779

J.M.Schoemaker, A.H.Brasnett and F.A.O.Marston 35 mM iodoacetamide (BDH Chemicals Ltd, Poole, UK) in sealed tubes under N2. Samples were incubated for 50 min at room temperature to allow reactions of iodoacetamide with protein thiol groups. For isolation of inclusion bodies, cells were lysed in the same buffer as above except SDS was replaced with 260 tg/ml lysozyme. This preparation was incubated in the presence of 1.0 mM phenylmethylsulfonyl fluoride for 10 min at 0°C, followed by addition of sodium deoxycholate to 0.1 % w/v and incubation for 5 min at 25°C. Cell extracts were finally incubated in the presence of 0.004% DNase for 30 min and then centrifuged at 12 000 g for 10 min. Intact inclusions in the pellet were washed twice in the same lysis buffer. Prior to SDS-polyacrylamide gel electrophoresis, cell extracts or washed inclusion preparations were heated at 95°C for several minutes in a Tris-SDS sample buffer, with or without 5% 2-mercapteothanol. SDS-polyacrylmaide gel electrophoresis was performed as described (Laemmli, 1970). To estimate the mol. wts. of proteins on Western blots, the following protein standards were used: phosphorylase b (97 400), bovine serum albumin (67 000), ovalbumin (43 000), carbonic anhydrase (30 000) and chymotrypsinogen (25 700).

Acknowledgements The authors wish to thank their colleagues at Celltech Ltd.: J.S.Emtage, P.A.Lowe, G.O.Humphreys and T.J.R.Harris for many helpful contributions to both the research and writing of this manuscript, and Julia Spragg for providing anti-prochymosin antibodies. We are also grateful to Robert Freedman for very valuable discussions and comments.

References Burnette,W.N. (1981) Anal. Biochem., 112, 195-203. Emtage,J.S., Angal,S., Doel,M.T., Harris,T.J.R., Jenkins,B., Lilley,G. and Lowe,P.A. (1983) Proc. Natl. Acad. Sci. USA, 80, 3671-3675. Foltmann,B., Pedersen,V.B., Jacobson,H., Kauffman,D. and Wybrandt,G. (1977) Proc. Natl. Acad. Sci. USA, 74, 2321-2324. Freedman,R.B. and Hillson,D.A. (1980) in Freedman,R.B. and Hawkins,H.C. (eds.), Enzymology of Post-translational Modification of Proteins, Vol. 1, Academic Press, London, pp. 157-212. Harris,T.J.R (1983) in Williamson,R. (ed.), Genetic Enineering 4, Academic Press, London, pp. 128-175. Harris,T.J.R. Lowe,P.A., Lyons,A., Thomas,P.G., Eaton,M.A.W., Millican,T.A., Patel,T.P., Bose,C.C., Carey,N.H. and Doel,M.T. (1982) Nucleic Acids Res., 10, 2177-2187. Klier,A., Fargette,F., Ribier,J. and Rapoport,G. (1982) EMBO J., 1, 791-799. Laemmli,U.K. (1970) Nature, 277, 680-685. Marston,F.A.O., Lowe,P.A., Doel,M.T., Schoemaker,J.M., White,S. and Angal,S. (1984) Biotechnology, 2, 800-804. Nishimori,K., Shimizu,N., Kawaguchi,Y. Hidaka,M., Uozumi,T. and Beppu,T. (1984) Gene, 29, 41-49. Schoemaker,J.M., Clark,J.M., Saukkonen,J.J. (1981) J. Gen. Microbiol., 123, 323-333. Shively,J.M. (1974) Annu. Rev. Microbiol., 28, 167-187. Towbin,H., Staehelin,T. and Gordon,J. (1979) Proc. Natl. Acad. Sci. USA, 76, 4350-4354. Weis,J.H, Enquist,L.W., Salstrom,J.S. and Watson,R.J. (1983) Nature, 302, 72-74. Wetzel,R. and Goeddel,D.V. (1982) in Gross,E. and Meinhofer,J. (eds.), The Peptides: Analysis, Synthesis, Biology, Academic Press, NY, pp. 1-58. Williams,D.C., Van Frank,R.M., Muth,W.L., Burnett,J.P. (1982) Science (Wash,), 215, 687-689. Yanofsky,C. (1981) Nature, 289, 751-758.

Received on 9 July 1984; revised on 31 December 1984

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