title: Cloning and Expression of Yak Active Chymosin in Pichia pastoris.
abstract: Rennet, a complex of enzymes found in the stomachs of ruminants, is an important component for cheese production. In our study, we described that yak chymosin gene recombinant Pichia pastoris strain could serve as a novel source for rennet production. Yaks total RNA was extracted from the abomasum of an unweaned yak. The yak preprochymosin, prochymosin, and chymosin genes from total RNA were isolated using gene specific primers based on cattle chymosin gene sequence respectively and analyzed their expression pattern byreal time-polymerase chain reaction. The result showed that the chymosin gene expression level of the sucking yaks was 11.45 times higher than one of adult yaks and yak chymosin belongs to Bovidae family in phylogenetic analysis. To express each, the preprochymosin, prochymosin, and chymosin genes were ligated into the expression vector pPICZαA, respectively, and were expressed in Pichia pastoris X33. The results showed that all the recombinant clones of P. pastoris containing the preprochymosin, prochymosin or chymosin genes could produce the active form of recombinant chymosin into the culture supernatant. Heterologous expressed prochymosin (14.55 Soxhlet unit/mL) had the highest enzyme activity of the three expressed chymosin enzymes. Therefore, we suggest that the yak chymosin gene recombinant Pichia pastoris strain could provide an alternative source of rennet production.
authors.affiliation: College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan 610041, China.|Key Laboratory of Animal Genetics & Breeding, State Ethnic Affairs Commission and Ministry of Education, Chengdu, Sichuan 610041, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Luo
authors.affiliation: College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan 610041, China.|Key Laboratory of Animal Genetics & Breeding, State Ethnic Affairs Commission and Ministry of Education, Chengdu, Sichuan 610041, China.
authors.forename: Wei Hua
authors.initials: WH
authors.lastname: Jiang
authors.affiliation: College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan 610041, China.|Key Laboratory of Animal Genetics & Breeding, State Ethnic Affairs Commission and Ministry of Education, Chengdu, Sichuan 610041, China.
authors.forename: Yuan Xiao
authors.initials: YX
authors.lastname: Yang
authors.affiliation: Institute of Qinghai-Tibetan Plateau, Southwest University for Nationalities, Chengdu, Sichuan 610041, China.
authors.forename: Jiang
authors.initials: J
authors.lastname: Li
authors.affiliation: College of Life Science and Technology, Southwest University for Nationalities, Chengdu, Sichuan 610041, China.|Key Laboratory of Animal Genetics & Breeding, State Ethnic Affairs Commission and Ministry of Education, Chengdu, Sichuan 610041, China.
authors.forename: Ming Feng
authors.initials: MF
authors.lastname: Jiang
keywords: Active Chymosin; Expression; Pichia pastoris; Yak