Expression of cloned calf prochymosin cDNA under control of the tryptophan promoter.
To increase yields of calf prochymosin (PC) produced in Escherichia coli, PC cDNA was cloned in a plasmid vector under control of the trp promoter. The hybrid plasmid pCR501 constructed for this purpose contains cDNA coding for PC (from the 5th Arg to the C-terminal Ile) fused to the N-terminal fragment of the trpE gene preceded by the trp promoter and attenuator region. E. coli C600 harboring this plasmid produces approx. 300 000 molecules of PC per cell. This is about a tenfold increase above the amount obtained using lacUV5 promoter [Nishimori et al., Gene 19 (1982) 337-344]. A similar plasmid, pCR601, which contains the same coding sequence fused to the trp promoter and N-terminal fragment of the trpL gene, directs the production of PC at the same rate as pCR501. In pCR601 the trp attenuator is deleted. Another plasmid, pCR701, in which construction of a sequence coding for fMet-PC cDNA that was aided by chemical synthesis, was placed under direct control of the trp promoter, produced PC at a much lower rate. Extracts prepared from all these bacterial transformants in the presence of urea showed distinct milk-clotting activity after renaturation and processing.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D012085:Chymosin / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004792:Enzyme Precursors / Q000032:analysis / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D009693:Nucleic Acid Hybridization; D009876:Operon; D010957:Plasmids; D014176:Protein Biosynthesis; D014158:Transcription, Genetic; D014364:Tryptophan / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics
D004792:Enzyme Precursors; D014364:Tryptophan; D004247:DNA; D004262:DNA Restriction Enzymes; D001616:beta-Galactosidase; C032443:prorennin; D012085:Chymosin