Recombinant calf prochymosin synthesized in E. coli was shown to accumulate in the form of insoluble inclusion bodies. Isolation of this aggregated material, combined with specific washing procedures, was the most significant stage of the purification protocol. Disruption of proteins in the inclusion bodies necessitated denaturation and renaturation. The method described can completely solubilize prochymosin. At this stage acidification produced active chymosin. Subsequently, ion-exchange chromatography produced highly purified prochymosin which, after acidification, yielded chymosin >99% pure.

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-cellulose 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.

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.

Two dodecadeoxynucleotides of defined sequence have been synthesised by phosphotriester methodology. They can be polymerised to give a double stranded DNA which codes, when read in the correct phase, for the repeating dipeptide poly(aspartyl-phenylalanine). This polymeric DNA has been cloned in E. coli K12 using as vector a plasmid having a controllable bacterial promoter upstream of the insertion site. Clones containing genes coding for up to 150 repeats of (aspartyl-phenylalanine) have been isolated and characterised. The polymeric inserts appear to be stable over many generations and are expressed in E. coli under the control of the bacterial promoter, to give a polymer of phenylalanine and aspartic acid which may be broken down enzymically to yield aspartyl-phenylalanine.

Conference Article| August 01 1978 The Ovalbumin Structural Gene in Genome and Messenger NORMAN H. CAREY; NORMAN H. CAREY 1Searle Research Laboratories, Lane End Road, High Wycombe, Bucks. HP12 4HL, U.K. Search for other works by this author on: This Site PubMed Google Scholar MICHAEL T. DOEL MICHAEL T. DOEL 2Searle Research Laboratories, Lane End Road, High Wycombe, Bucks. HP12 4HL, U.K. Search for other works by this author on: This Site PubMed Google Scholar Author and article information Publisher: Portland Press Ltd Online ISSN: 1470-8752 Print ISSN: 0300-5127 © 1978 Biochemical Society1978 Biochem Soc Trans (1978) 6 (4): 746–747. https://doi.org/10.1042/bst0060746 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn Email Cite Icon Cite Get Permissions Citation NORMAN H. CAREY, MICHAEL T. DOEL; The Ovalbumin Structural Gene in Genome and Messenger. Biochem Soc Trans 1 August 1978; 6 (4): 746–747. doi: https://doi.org/10.1042/bst0060746 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1978 Biochemical Society1978 Article PDF first page preview Close Modal You do not currently have access to this content.

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.

We present evidence that the poly(A) sequence at the 3′ end of ovalbumin mRNA has an effect on its translational efficiency in a reticulocyte lysate cell-free system. Polynucleotide phosphorylase has been used to remove selectively the poly(A) while leaving the rest of the molecule intact. It is shown that the stability of the mRNA in a cell-free system is not appreciably affected by this procedure.

The sequence of 75 bases adjacent to the 3′ terminal poly(A) region of chicken ovalbumin messenger RNA was established by copying into 32P-labelled DNA and sequencing the DNA directly. The sequence immediately adjacent to the poly(A) differs from a mouse MOPC 21 immunoglobulin light chain mRNA; this confirms that there is no obligatory sequence homology here. But, about 20 residues internal to the poly(A), the sequence (5′)A-A-U-A-A-A-(3′) is conserved in the four different mRNAs, rabbit α- and β-globin, immunoglobulin light chain and ovalbumin, although its significance is unknown.