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A gene for calf prochymosin (prorennin) has been reconstructed from chemically synthesized oligodeoxyribonucleotides and cloned DNA copies of preprochymosin mRNA. This gene has been inserted into a bacterial expression plasmid containing the Escherichia coli tryptophan promoter and a bacterial ribosome binding site. Induction of transcription from the tryptophan promoter results in prochymosin synthesis at a level of up to 5% of total protein. The enzyme has been purified from bacteria by extraction with urea and chromatography on DEAE-celiulose and converted to enzymatically active chymosin by acidification and neutralization. Bacterially produced chymosin is as effective in clotting milk as the natural enzyme isolated from calf stomach.Chymosin (rennin) is an aspartyl proteinase found in the fourth stomach of the unweaned calf, where it is responsible for limited proteolysis of K-casein in milk (1), resulting in clotting (2). Full-length cDNA copies of the mRNA for chymosin have recently been cloned and their sequences have been determined (3)(4)(5). The primary translation product of the mRNA is a precursor protein, preprochymosin, that has a 16-amino acid leader peptide. This signal sequence is removed to produce the zymogen prochymosin, which in turn is converted to active chymosin under the acid conditions of the stomach by removal of the 42-amino acid propeptide from the NH2-terminus (6).Recombinant plasmids containing the preprochymosin coding sequence provided a choice of genes for expression in Escherichia coli-i.e., preprochymosin, prochymosin, or chymosin. Preprochymosin and chymosin were ruled out for the following reasons. For preprochymosin, the E. coli cell would have to recognize the eukaryotic signal peptide and process it accurately to prochymosin, which would then be found in the periplasmic space. Despite the fact that E. coli processes the rat preproinsulin gene in this manner (7), it does not, apparently, process preinterferon-a (8), preinterferon-f3 (9), or pregrowth hormone (10) in the same way. Direct expression of the chymosin gene would be expected to lead, by analogy with human growth hormone (11), to production of chymosin containing an additional methionine residue at the NH2 terminus ([Met]chymosin). Apart from possible deleterious effects of the production of active chymosin in E. coli, there was also the possibility that an NH2-terminal methionine would alter the activity or tertiary structure of the enzyme. The expression of prochymosin, on the other hand, offered positive advantages. First, the 42-amino acid propeptide can be removed in vitro by acidification followed by incubation at pH 6 (12) and, assuming that a [Met]prochymosin could be cleaved in similar fashion, the product would be authentic chymosin rather than [Met]chymosin. Second, since preliminary experiments indicated that the majority of E. coli proteins (about 90%) precipitated under the acidic chymosinThe publication costs of this article were defrayed in part by page charge payment. This article must ...

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Chicken DNA has been digested with restriction enzymes and the size distribution of the DNA fragments containing ovalbumin specific sequences has been examined after separation of the fragments on agarose gels and transfer to nitrocellulose sheets. Hybridisation with terminally 32P-labelled ovalbumin mRNA fragments or with RNA populations transcribed from the DNA of a hybrid plasmid containing ovalbumin sequences was used to locate the DNA fragments coding for ovalbumin. Digestion with enzymes which do not cut within the portion of the ovalbumin gene synthesised from ovalbumin messenger RNA in vitro has shown the presence of several defined fragments carrying ovalbumin specific sequences. Possible explanations of these observations are discussed.

DNA complementary to calf stomach mRNA has been synthesised and inserted into the Pst1 site of pAT153 by G-C tailing. Clones containing sequences coding for prochymosin were recognised by colony hybridisation with cDNA extended from a chemically synthesised oligodeoxynucleotide primer, the sequence of which was predicted from the published amino acid sequence of calf prochymosin. Two clones were identified which together contained a complete copy of prochymosin mRNA. The nucleotide sequence is in substantial agreement with the reported amino acid sequence of prochymosin and shows that this protein has a mol.wt. of 40431 and chymosin a mol.wt. of 35612. The sequence also indicates that prochymosin is synthesised as a precursor molecule, preprochymosin, having a 16 amino acid hydrophobic leader sequence analogous to that reported for other secreted proteins.