Production of full length and splicing form of chymosin using pETexpression system in E-coli and investigation its enzyme activity and preplasmic secretion

Introduction: Chymosin (Rennin EC 3.4.23.4) is an aspartyl proteinas (the major proteolyticenzyme in the fourth stomach of the unweaned calf) that is formed by proteolytic activation fromzymogene prochymosin. The aim of his study was to produce the full length and splicing form ofchymosin using pETexpression system in E-coli and to assay the activity of expressed enzyme andpreplasmic secretion.Materials and Methods: The sense primer F-prochy(+) (5′-ggggccatgGCTGAGATCACCAGGA)including NCOI restriction site). The anti sense R-prochy(-) (5′-gggcggccgcGATGGCTTTGGCCAGC -3′) hybridizing to the C-terminal end of calf preprocymosincDNA and contains an additional NotI restriction site at its 5′-end . The cells were disrupted bysonication and proteins were purified by using Ni-NTA beads from Qiagen under native conditional.The preprochymosin and AS6 preprochymosin were activated at pH 4.7. The enzyme solutions werediluted 20-fold with 50 mM phosphate buffer .Results: Sequencing data analysis showed that the exon six has been spliced out and, therefore thegene product is 114 bp shorter in length, both chymosin forms were expressed together in E.coli.Under the same expression conditions, at least AS6 preprochymosin was produced 7-fold highexpression in comparison to a full-length recombinant chymosin. Following acid activation andneutralization, the purified fractions were tested in a qualitative milk clotting assay. The clottingactivity of preprochymosin and exon6-less preprochymosin were comparable.Conclusion: High expression of this alternatively expressed transcript in bacteria, and properfolding of the AS6 chymosin protein molecule in the absence of exon six are the two most importantaspects distinguished in this research.