High-level Expression And Purification Of Codon Optimized Proteinase K In Pichia Pastoris

Proteinase K is an extracellular serine endoproteinase synthesized by the fungus Triitirachium album Limber.It has attacted intensive research interest from the academic,industrial and agricultural communities.Proteinase K is obtained commercially in large amounts by fermentation of Triitirachium album Limber. However, Triitirachium album Limber is a slowly growing fungus which only secretes small amounts of proteases into the medium.Moreover,it is unsuitable for fermentation on a large scale.In order to improve expression level of proteinase K and simplify the purification process, the gene of proreinase K was first optimized and synthesized based on the preferred codon usage of Pichia pastoris. Otherwise,the gene was fused in frame with a string of nucleotides encoding six histidine residues(His6-Tag) and then introduced it into P.pastoris GS115. Proteinase K was secreted as a secretory form in P. pastoris GS115, which simplified the purification process.Before fermentation, the expression conditions such as the supplement of methanol, fermentation temperature, pH and the induction time was optimized. We also compared the effect of three methods for purifying the proteinase K and select the best one.The result showed that, we constructed the yeast expression vector of proteinase K successfully and the proteinase K can be secreted by the P. pastoris GS115as a soluble form. The optimal fermentation conditions were supplement of methanol0.75%, fermentation temperature25℃, pH7.0. Under the optimized fermentation condition, the final yield of proteinase K obtained was about2.2g per liter of culture. By comparing different methods for purifying the proteinase K,the method that the centrifugal supernatant from fermentation broth containing proteinase K was purified by Ni-NTA affinity chromatograph directly is the best one.