Effects Of ?-factor Signal Peptides And Its N-glycosylation On Secretion Of Heterologous Proteins In Pichia Pastoris

Pichia pastoris expression system has been a very powerful tool to express heterologous proteins because it possesses the advantage of eukaryotes and prokaryotes.Up to now,more than 1000 heterologous proteins have been successfully expressed in the expression system.The expression levels of heterologous proteins are one of the most important factors as a successful expression system.Although some heterologous proteins are reported for highly secreted expression in Pichia pastoris,most proteins can only be expressed in a very low level to acquire the mg/L in Pichia pastoris.Therefore,how to find a universal method to improve the secreted expression of heterologous proteins in Pichia pastoris has an important significance for the improvement of Pichia pastoris expression system.Signal peptides is a very important prerequisite for secreted proteins to enter the secretory pathway and have a significant influence on the secreted expression of proteins.In this study,enhanced green fluorescent protein(EGFP)was used as a reporter to study the effects of three kinds of ?-factor signal peptides(S.cescerevisiae,K.pastoris and K.phaffii)and N-glycosylation on secreted expression for heterologous protein in Pichia pastoris.The results showed that the ?-factor signal peptide of S.cerevisiae could well guide EGFP to secret into the medium in Pichia pastoris,while the signal peptides of K.pastoris and K.phaffii could not do as well as ?-factor signal peptide of S.cerevisiae.They were lower efficient in EGFP secretion compared with S.cescerevisiae's.The expression levels for EGFP using K.pastoris and K.phaffii ?-factor signal peptides were 98.2 times and 13.5 times lower than that using S.cescerevisiae's ?-factor signal peptide,respectively.To reveal the causes of low expression levels in K.pastoris and K.phaffii ?-factor signal peptides,protein retention,protein degradation,gene integration copy number,gene transcription and codon preference were analyzed.However,they were all not the reasons that caused inefficiency in the secreted expression of EGFP.The potential N-glycosylation sites in ?-factor signal peptides of S.cescerevisiae,K.pastoris and K.phaffii was analyzed,which discovered that there are three N-glycosylation sites on the pro-peptide in S.cescerevisiae ?-factor signal peptide,and there is a lack of N-glycosylation sites in K.pastoris and K.phaffii ?-factor signal peptides.When the amino acid Asn(N)of the all three Asn-X-Ser/Thr motifs on the pro-peptides of S.cescerevisiae ?-factor were mutated into Gln(Q),which making them unable to be glycosylated,the expression levels of EGFP were decreased by nearly 50.3%.The introduction of a N-glycosylation site on the pro-peptides of K.pastoris and K.phaffii ?-factors could significantly increased the expression levels by 31.5-fold and 5.2-fold,respectively.These results indicated that the glycosylation modification in the pro-peptide of ?-factor contributed to the secreted expression of the heterology protein in Pichia pastoris.The lack of glycosylation in ?-factor signal peptides could be the reason which ?-factor signal peptides of K.pastoris and K.phaffii do not work well in guiding the secretion of heterology proteins in Pichia pastoris.Furthermore,the ?-factor signal peptide of S.cescerevisiae was used as a model to study the effect of the number and position of N-glycosylation sites in the pro-peptide on the secreted expression in Pichia pastoris.The results showed that the number and position of N-glycosylation sites in the pro-peptide had no effect on the expression levels of the reporter EGFP in Pichia pastoris.The study provides a technical method to improve the expression levels of heterologous proteins in Pichia pastoris,and also establishes a good early stage for the research on mechanism of the effect of Nglycosylation on secreted expression of proteins in Pichia pastors.At the same time,the development of ?-factor signal peptides of K.pastoris and K.phaffii in the study also offers more options for expression of heterologous proteins in Pichia pastoris.