Expression of calf prochymosin in Saccharomyces cerevisiae.
A yeast strain which synthesizes activatable calf prochymosin (also known as prorennin) has been constructed by transformation with a vector carrying the methionyl-prochymosin coding sequence attached to efficient yeast transcriptional promoter and terminator sequences. Cloned preprochymosin cDNA was altered by restriction endonuclease cleavage and addition of a synthetic oligonucleotide to yield a DNA sequence encoding methionyl-prochymosin. This methionyl-prochymosin gene was ligated to a yeast chromosomal fragment containing the GAL1 promoter, and the construction was placed in an Escherichia coli-Saccharomyces cerevisiae shuttle vector with or without a transcriptional terminator DNA fragment from the yeast SUC2 gene. In yeast the two constructions result in equal amounts of prochymosin protein and mRNA. The prochymosin from yeast is activatable to chymosin by incubation at low pH and exhibits milk-clotting activity indistinguishable from calf chymosin.
D000818:Animals; D002417:Cattle; D012085:Chymosin / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D013045:Species Specificity; D014158:Transcription, Genetic
D004274:DNA, Recombinant; D004792:Enzyme Precursors; D012333:RNA, Messenger; C032443:prorennin; D012085:Chymosin