PSX-B-12 Сreation of a pichia pastoris strain: A producer of recombinant peptidase M9 from Aeromonas salmonicida

Abstract

Abstract Proteases are produced by a wide variety of microorganisms and have a wide range of applications. Peptidase M9 from Aeromonas salmonicida is a 915 amino acid residue protein. Earlier, we cloned the 864 bp C end of the gene. (col2 gene) encoding the actual peptidase domain of 238 aa in the vector plasmid pPic9K. It was demonstrated that the recombinant Pichia pastoris strain, created on the basis of this plasmid, has a specific enzymatic activity against collagen. The aim of this work was to further optimize the expression of the col2 gene of the M9 peptidase domain from Aeromonas salmonicida in Pichia pastoris, isolation, purification, and characterization of the protein.First, the col2 gene was optimized based on the use of P. pastoris codons. Secondly, a study of collagenase activity with 4 different signaling sequences was carried out. It was shown that the strain for which the collagenase gene had the P. pastoris alpha peptide as a signal sequence was shown to have the maximum activity. One of the approaches leading to an increase in the yield of the target protein is the cloning of the gene encoding the chaperone proteins. We have created strains containing, in addition to the collagenase gene, also the pdi and hac chaperone genes. It was shown that the collagenase activity in the strain containing the pdi chaperone gene was 2–3 times higher than in the strain containing only the col2 gene. So, the Pichia pastoris strain was created - the producer of the recombinant peptidase M9 from Aeromonas salmonicida, the target protein was isolated and characterized.Research conducted as part of the state assignment of Gorbatov Federal Research Center for Food Systems of Russian Academy of Sciences 0437-2019-0001.