Study On Signal Peptides That Efficiently Guide Exogenous Protein Secretion In Pichia Pastoris

As one of the most popular expression systems,Pichia pastoris expression system has been widely used in the fields of food,agriculture,and chemical pharmacy.Exogenous proteins secreted from this system require the guidance of signal peptides into the endoplasmic reticulum(ER)for their modification and maturation,and therefore signal peptides are essential for the efficient secretion of exogenous proteins in Pichia pastoris.With the completion of the genome sequencing of fungal microorganisms and Pichia pastoris,more ?-factor signal peptides from fungi and endogenous signal peptides from Pichia pastoris have been discovered.Up to now,no article has systematically evaluated the function of these signal peptides in secretion of exogenous proteins in Pichia pastoris system.In this study,the ?-factor signal peptides of different species and the endogenous signal peptides of Pichia pastoris were fully evaluated for their secretory efficiency in the expression of exogenous protein using EGFP as a reporter.A total of 40 ?-factor signal peptides of different species were found by searching the NCBI database.DNA sequences of 40 ?-factor signal peptides were cloned respectively into the reporter vector p PPIC9K-EGFP containing EGFP gene,and the recombinant vectors were integrated into GS115 to examine the expression of EGFP.The results showed that the signal peptide AF-27 could express more efficiently EGFP than that from S.cerevisiae(about 1.2 times).The secretory efficiency of ?-factor signal peptides from 18 species was equivalent to that of the ?-factor signal peptide from S.cerevisiae(about 0.8to 1 time).The ?-factor signal peptides from another18 species did not show high secretory efficiency.Only the signal peptides AF-31 and AF-33 could not secret EGFP.The results indicated that ?-factor signal peptides of different species are universally applicable in Pichia pastoris,also suggesting the conservation of the mechanism based on the ?-factor signal secretion.The phylogenetic tree was constructed based on the sequence of ?-factor signal peptides,which was consistent with the phylogenetic relationships described in many literatures,indicating that the sequence of ?-factor signal peptides can be used to construct the phylogenetic tree to judge the relationships of species.According to the signal peptide D-Score,32 endogenous signal peptides were selected using 0.95 as the limit value.The DNA sequences of the 32 signal peptides were cloned into the reporter vector p PPIC9K-EGFP containing EGFP,and the recombinant vectors were integrated into GS115 to examine the expression of EGFP.The results showed that there are 24 endogenous signal peptides that could guide the secretion of EGFP.9 signal peptides could efficiently secret EGFP.This study can provide options of signal peptides for the highly secretory expression of exogenous proteins in Pichia pastoris and the modification.At the same time the signal peptides screened in this study can also be used to explore the molecular mechanism of secretary pathway in Pichia pastoris.Therefore,this work is valuable to further investigate the ?-factor signal peptides of different species and the signal peptides of Pichia pastoris.