The Study Of Furin Site Processing In Pichia Pastoris

In the biosphere,a large number of proteins are synthesized in the form of precursor.They are mainly composed of three parts: signal peptide,precursor peptide and mature peptide.The leading peptide plays an important role in the correct folding of mature peptide.furin,a protein invertase,is the key to remove precursor peptide from mature peptide and then the mature peptide can achieve physiological activity.furin protein invertase has various substrates,most of which are pharmaceutical protein candidates or protein drugs.But no study shows that when the precursor peptide and mature peptide are expressed in Pichia pastoris at the same time,whether the furin sites between the propeptide and mature peptide can be cut accurately,which can make recombinant protein obtaining physiological activity in Pichia pastoris.So we try to do a thorough study in this direction.This study used EGFP as a reporter protein for furin sites digestion detection and the furin sites between propeptide alpha-factor and mature peptide is RSRR,RSIR and RSKR.After construction of EGFP reporter vector containing furin sites,the reporter vector transferred into Pichia pastoris.Then in next step,we found that the processing efficiency of RSRR,RSIR and RSKR is 29.7%,29% and 49.3%respectively in GS115.It means that furin locus can be processed in Pichia pastoris,but the efficiency is low.In mammals,we found that P4 sites with P1 dependence in processing furin sites.In order to verifying whether the Pichia pastoris processing furin locus also has sequence dependence,we constructed the expression vector containing multiple furin mutation type.The experimental results show that in Pichia pastoris mutation RSII,ISIR is no longer to be processed,and the processing efficiency of RXKI,IXKR mutation sites were decreased by 79% and 62%,relative to RXKR sites.Then processing efficiency of RXRR point mutations RXRI and IXRR decreased by 75% and 68%.These result proved that the process of furin loci in Pichia pastoris have the characteristic sequence dependence.Mechanism of furin locus digestion in Pichia pastoris was explored through gene knockout.We found Kex2 participates in furin loci processing in Pichia pastoris.While knockdown of Yps1,there is no any processing within furin sites,which prove that Kex2 is responsible for the furin loci process in Pichia pastoris.In order to promoting the efficient expression of exogenous precursor protein in Pichia pastoris,we have tried to overexpress Kex2 or furin.We found that in the overexpression of Kex2 strains,the processing efficiency for RSRR,RSIR and RSKR loci were increased by 33.8%,56.2% and 38.7% respectively,while in overexpression of furin strains,the processing efficiency of RSRR,RSIR and RSKR locus increased 43.9%,48.3% and 9.9%respectively.Two kinds of engineering strains can improve the efficiency of furin site processing.This research is about exploring the Pichia pastoris processing mechanism of the furin locus,which explain the digestion of precursor protein containing furin sites in Pichia pastoris.Then through overexpression of furin or Kex2 in Pichia pastoris,it does solve the processing problem of exogenous precursor protein expressed in Pichia pastoris.