Translation of preprochymosin in vitro. Evidence for folding of prochymosin to the native conformation.
1. The cDNA coding for preprochymosin has been sub-cloned into the transcription/translation vector pGEM-3Z, the T7 promoter used to transcribe the gene and the product expressed in an 'in vitro' cell-free system comprising rabbit reticulocyte lysate and dog pancreatic microsomes. 2. Translations in various conditions, and analyses of the translation product in reducing and non-reducing conditions, indicate that oxidizing translation conditions and the cleavage of the N-terminal 'pre-' sequence are essential for generation of a disulphide-bonded translation product. 3. The disulphide-bonded translation product was resistant to proteinases, as expected for a translation product segregated within microsomal vesicles; in the presence of detergent to solubilize the membranes, the product was not readily susceptible to proteolysis, and was converted to a proteinase-resistant core fragment. 4. Segregated prochymosin, synthesized in reducing conditions, was completely degraded by proteinases under similar conditions. 5. Proteinase treatment of purified recombinant prochymosin gave rise to a proteinase-resistant fragment of similar Mr, suggesting that the disulphide-bonded product of translation in vitro was correctly folded. 6. The translocated, disulphide-bonded and folded prochymosin could be converted into pseudochymosin at pH 2.0, and addition of chymosin to the activation mixture resulted in increased pseudochymosin production.
Sheikh
A
A
Biological Laboratory, University of Kent, Canterbury, U.K.
Freedman
R B
RB
D000818:Animals; D002417:Cattle; D012085:Chymosin / Q000235:genetics*; D003001:Cloning, Molecular; D004285:Dogs; D004792:Enzyme Precursors / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D008861:Microsomes / Q000378:metabolism; D010179:Pancreas / Q000378:metabolism; D010957:Plasmids; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011499:Protein Processing, Post-Translational; D051381:Rats; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D014158:Transcription, Genetic
D004792:Enzyme Precursors; C034675:preprorennin; C032443:prorennin; D012085:Chymosin