Recombinant Protein Production In Pichia Pastoris Expression System

The Pichia pastoris is one of the most successful foreign protein expression system until now. It offers economy, ease of manipulation, the ability to peform complex post-translational modifications, and high expression levels. It does not have the endotoxin problem associated with bacteria nor the viral contamination problem of recombinant proteins produced in mammalian cell culture. To date many heterologous proteins have been expressed in Pichia pastoris successfully. Several recombinant were produced in the expression system.1. consensus interferon mutantAn artificial gene for consensus interferon mutant was synthesized by using favored codons of the yeast Pichia pastoris. The gene was cloned into the secretory expression vector, then linearized and transformed into GS115 for expression. Recombinant protein level was 112mg/L in fermenter culture. The purity of recombinant protein reached over 98% through ultrafiltrated and three steps chromatography. The specific activity of protein was 6×10~8 antiviral units/mg.2. IFN β-1aFFN β-1a gene was synthesized, then inserted into the expression vector and transformed into GS115. Induced by methanol, the expression level of culture supernatant was 2×10~5IU/mL of antiviral activity by a cytopathic effect inhibition assay.3. Tissue Plasminogen Activator mutant rPAmThe yeast expression vector of rPAm was constructed, and rPAm was expressed in culture supernatant, the expression level was 1.6mg/L.4. Thr6-PDGF-BBThe expression vector of Thr6-PDGF-BB was constructed, and the elegant transformant was selected. The expression level of the recombinant protein was 320mg/L in high-density fermentation. An optimized purification process wasdeveloped, the purity of Thr6-PDGF- reached over 99% through ultrafiltrated and three steps chromatography.