Proc. Nati Acad. Sci. USA Vol. 80, pp. 3671-3675, June 1983 Biochemistry Synthesis of calf prochymosin (prorennin) in Escherichia coli (synthetic oligonucleotide/gene expression/industrial enzyme) J. S. EMTAGE*, S. ANGALt, M. T. DOELt, T. J. R. HARRISt, B. JENKINS*, G. LILLEYt, AND P. A. LOWEt Departments of *Molecular Genetics, tMolecular Biology, and tFermentation Development, Celltech Limited, 250 Bath Road, Slough SL1 4DY, Berkshire, United Kingdom Communicated by Sydney Brenner, March 23, 1983 ABSTRACT A gene for calf prochymosin (prorennin) has been maturation conditions and remained insoluble on neutraliza- reconstructed from chemically synthesized oligodeoxyribonucleo- tion, it was possible that purification as well as activation could tides and cloned DNA copies of preprochymosin mRNA. This gene be achieved. has been inserted into a bacterial expression plasmid containing We describe here the construction of E. coli plasmids de- the Escherichia coli tryptophan promoter and a bacterial ribo- signed to express the prochymosin gene from the trp promoter some binding site. Induction oftranscription from the tryptophan and the isolation and conversion of this prochymosin to en- promoter results in prochymosin synthesis at a level of up to 5% active of total protein. The enzyme has been purified from bacteria by zymatically chymosin. extraction with urea and chromatography on DEAE-celiulose and MATERIALS AND METHODS converted to enzymatically active chymosin by acidification and neutralization. Bacterially produced chymosin is as effective in Materials. DNase I, pepstatin A, and phenylmethylsulfonyl clotting milk as the natural enzyme isolated from calf stomach. fluoride were obtained from Sigma. Calf prochymosin (Mr 40,431) and chymosin (Mr 35,612) were purified from stomachs Chymosin (rennin) is an aspartyl proteinase found in the fourth from 1-day-old calves (1). Rabbit antiprochymosin antiserum stomach of the unweaned calf, where it is responsible for lim- was provided by the National Institute of Medical Research (Mill ited proteolysis of K-casein in milk (1), resulting in clotting (2). Hill, London). Restriction enzymes and T4 DNA ligase were Full-length cDNA copies of the mRNA for chymosin have re- purchased from New England BioLabs, polynucleotide kinase cently been cloned and their sequences have been determined was from Bethesda Research Laboratories, and calf intestinal (3-5). The primary translation product of the mRNA is a pre- alkaline phosphatase and S1 nuclease were from Boehringer cursor protein, preprochymosin, that has a 16-amino acid leader Mannheim. Restriction enzymes were used under conditions peptide. This signal sequence is removed to produce the zymo- recommended by the supplier. Oligodeoxyribonucleotides R29 gen prochymosin, which in turn is converted to active chymosin (5'-phosphate-G-A-T-C-C-G-A-G-T-G-A-T-T-T-C-A-G-C) and under the acid conditions of the stomach by removal of the 42- R30 (5'-hydroxy-G-C-T-G-A-A-A-T-C-A-C-T-C-G) were syn- amino acid propeptide from the NH2-terminus (6). thesized by the phosphotriester procedure (13) using preferred Recombinant plasmids containing the preprochymosin cod- E. coli codons (14). ing sequence provided a choice of genes for expression in Esch- Bacterial Strains. E. coli K-12 strain HB101 (15) containing erichia coli-i.e., preprochymosin, prochymosin, or chymosin. the plasmids pCT54 or pCT70 were grown at 37°C in M9 me- Preprochymosin and chymosin were ruled out for the following dium (16) supplemented with glucose, all amino acids except reasons. For preprochymosin, the E. coli cell would have to rec- tryptophan (and methionine when labeling proteins with [3S]- ognize the eukaryotic signal peptide and process it accurately methionine), and 100 ug of carbenicillin per ml. Large quan- to prochymosin, which would then be found in the periplasmic tities of cells were prepared by growth for 10 hr to an OD6oo space. Despite the fact that E. coli processes the rat preproinsu- of 10 (1 x 109 cells per ml) and harvested by centrifugation or lin gene in this manner (7), it does not, apparently, process ultrafiltration. preinterferon-a (8), preinterferon-f3 (9), or pregrowth hormone Assay of Chymosin Milk-Clotting Activity. Prochymosin from (10) in the same way. Direct expression of the chymosin gene a calf or bacterial source was converted to catalytically active would be expected to lead, by analogy with human growth hor- chymosin by acidification/neutralization (activation) immedi- mone (11), to production of chymosin containing an additional ately prior to assay. One-milliliter samples were incubated at methionine residue at the NH2 terminus ([Met]chymosin). Apart pH 2.0 for 15 min at room temperature and then the pH was from possible deleterious effects of the production of active adjusted to 6.3 and incubation was continued for 1 hr. Insoluble chymosin in E. coli, there was also the possibility that an NH2- proteins were removed by centrifugation for 1 min in a bench terminal methionine would alter the activity or tertiary struc- centrifuge and samples of the supernatant were taken for assay. ture of the enzyme. The expression of prochymosin, on the other A simple microtiter plate assay was developed for the mea- hand, offered positive advantages. First, the 42-amino acid pro- surement of milk-clotting activity. The final assay mixture (100 peptide can be removed in vitro by acidification followed by ,ul per well) was 12% (wt/vol) dried skim milk/20 mM CaCl2/ incubation at pH 6 (12) and, assuming that a [Met]prochymosin 25 mM phosphate buffer, pH 6.3, and chymosin was incubated could be cleaved in similar fashion, the product would be au- at 37°C for 15 min. The plate was inverted and any unclotted thentic chymosin rather than [Met]chymosin. Second, since milk was allowed to drain over absorbent paper prior to pho- preliminary experiments indicated that the majority of E. coli tography of the wells containing clotted milk. The assay was proteins (about 90%) precipitated under the acidic chymosin capable of detecting approximately 0.02 ,g of chymosin al- though the sensitivity could be increased by prolonged incu- The publication costs of this article were defrayed in part by page charge bation. payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: bp, base pair(s). 3671 Downloaded by guest on October 1, 2021 3672 Biochemistry: Emtage et al. Proc. Natl. Acad. Sci. USA 80 (1983) Purification of Chymosin from E. coli. Cells [100 g (wet ification were removed by centrifugation and pure chymosin weight)] were lysed with lysozyme/sodium deoxycholate (17) was stored at -20'C. Further details of the isolation of chy- and treated with DNase (18). The resulting suspension was cen- mosin from E. coli will appear elsewhere. trifuged at 10,000 x g for 30 min at 4°C, the pellet formed was suspended in 300 ml of 50 mM Tris HCI, pH 8/0.1 M NaCI/ 1 mM EDTA and recentrifuged as above. Washed cell debris RESULTS AND DISCUSSION was dissolved in 200 ml of 9 M urea (freshly deionized)/50 mM Construction of the Prochymosin Gene. The approach used Tris HCI, pH 8/0.5 M NaCI/1 mM EDTA and dialyzed over- for the bacterial synthesis of [Met]prochymosin was to recon- night at 40C against 5 liters of 50 mM Tris'HCI, pH 8/0.5 M struct a gene in vitro from restriction fragments of a cDNA clone NaCI/1 mM EDTA/10% (vol/vol) glycerol. The suspension was and then use synthetic DNA fragments for insertion into a bac- centrifuged as above, and the supernatant diluted 1:4 with 0.01 terial plasmid immediately downstream from the strong E. coli M Tris HCI, pH 8/1 mM EDTA/10% (vol/vol) glycerol and trp promoter, a functional ribosome binding site and ATG. As applied to a 200-ml column (4.7 x 15 cm) of DEAE-cellulose shown in Fig. 1A, a BamHI restriction site is conveniently lo- previously washed and equilibrated in 0.01 M Tris HCI, pH 8/ cated between codons 5 and 6 of the prochymosin sequence. 1 mM EDTA/10% (vol/vol) glycerol. Adsorbed proteins were Two oligodeoxyribonucleotides were designed to restore co- eluted with a linear gradient of NaCl (0.05-0.5 M) in equili- dons 1-6 and produce a blunt 5' end with a 5'-OH group. These bration buffer. Prochymosin-containing fractions were de- oligomers were hybridized and ligated to the 474-base-pair (bp) tected by assaying activated samples for milk-clotting activity BamHI fragment from plasmid 118 (3) and the resulting DNA and by NaDodSO4/polyacrylamide gel electrophoresis of frac- was cleaved with EcoRI to produce a 478-bp DNA fragment tions across the profile. The enzyme eluted reproducibly as a coding for the 5' end of prochymosin. The 3' end of the pro- single peak at a NaCl concentration of 0.25 M. Appropriate pro- chymosin gene was isolated as an EcoBJ/HindIII fragment (Fig. chymosin fractions with a purity at this stage of approximately 1B) of =800 bp from plasmid C5. The HindIll cohesive end 50% were pooled and activated as described above to generate was dephosphorylated by calf intestine alkaline phosphatase chymosin. The insoluble protein impurities generated by acid- during isolation of the fragment to prevent unwanted ligation .AAGCTTGGAATTCATTGATCAATCGATACCCTT. .3' \. /. .T TCGAACCT TAA@4ACTAGT TAGCTATG .5' B EB H EB B H Cla1 - 118 . I I C5 I L I I I.i. I E E trp Po pre pro 1 Bam HI 1 Hind 111 2 Ligate R29/R30 2 Phosphatase Bcl1 Sal 1 3 EcoR 1 3 EcoR1 B I amp pCT54 I Ligate, kinase 1 EcoR 1 1 Hind 111 2 Isolate from gel B 2 S1 nuclease 2 Sal 1, isolate B 5'-OP, I TCGA-OP-5' 3 Sall, isolate A+ CATTGATCA ....