Studies On Cloning, Codon Optimization Of The Swine Pepsinongen A Gene And Expression In Pichia Pastoris

Aspartic acid proteases, known as acidic proteases, can catalyze the hydrolysis of protein under the acid condition, and is one of the most important industrial enzymes. Aspartic acid proteases are widely applied in the fields of feed additive, vintage, food, leather and medicine. Porcine pepsin A, which is the important model for the study of structure-function relationship of protein, belongs to the family of aspartic protease. In practice, the application of the acidic proteases suffers enormous limit, because the available commercial acidic proteases are instability and low production. Using gene engineering to study the expression of pepsin will supply a new way for solving these questions in this paper.Firstly, the primers were designed according to the sequence J04601 in Genebank. And then the swine pepsinogen A cDNA, which included an ORF of 1158 bp nucleotides, encoded 385 amino acids, was cloned by RT-PCR from the Hubei White swine. The N-terminal region of 385 amino acids, contained a signal peptide consist of fifteen amino acids and the mature protein consist of 370 amino acids. The result showed that identical of the nucleotide and amino acid was 99.3% and 98.7%, respectively, compared with J04601. This gene was submitted to NCBI Genebank and the accession number is EF108301.Secondly, the recombinant plasmid of pPIC3.5K-pA was constructed and transformed into Pichia pastoris GS115, based on the cloning and sequence analysis of pepsinogen A. Two over-expression recombinants were screened by the experiment of hemaleucin digestion and the method of casein-agar plate. Effects of different pH, different inductive time, different inductive concentration and methanol inductive concentration on the expression yield were studied. The results indicated that the yield of recombinant pepsinogen was able to achieve 56 mg/L, when the pH value was to keep 7, to induct 72 h, to choose induction concentration OD600 to 2, to keep the methanol induction concentration to 1%,which was 1.7 times than optimization. The recombinant pepsin had natural biologic activity by the detection of the western blotting.Thirdly, based on the codon bias of Pichia pastoris and used SMD technology, optimization of the rare codons, which were scattered at C- and N-terminal region and could influence the yield of recombinant, was directed successfully. The further research is expected to raise the yield of recombinant yeast.