Construction Of Novel Pichia Pastoris Cell-Surface Display Systems And Research Of Their Functions

With the application of recombinant DNA technology,cell-surface display systems have been developed in various microorganisms,such as in phage,in bacteria and especially in Saccharomyces cerevisiae.Microbial cell-surface display has been widely used in biotechnological research and industrial applications,including: identifying protein-protein interactions,displaying polypeptide libraries as selection devices,mapping functional protein epitopes,immobilizing proteins and enzymes as whole cell biocatalysts,producing antibodies and live vaccines,producing bioadsorbents for the removal of harmful chemicals and heavy metals,et al.There are obvious disadvantages in the present microbial cell-surface display systems.In phase-display system,when larger-sized polypeptides were fused to the major coat protein of the phage,the hybrids were not readily incorporated into the phage particle;In Gram-negative display systems,both translocation through the cytoplasmic membrane and correct integration into the outer membrane may affect the structure and function of the proteins.In Gram-positive bacteria display systems,the lower frequency of transformation restricts the creation of large combinatorial protein libraries.Both the Gram-negative display systems and Gram-positive bacteria display systems cann't fold and glycosylate the eukaryotic protein.In S.cerevisiae display system,secreted proteins are hyperglycosylated and have 50-150 mannoses per N-glycan,which may affect the activity of enzyme.In addition,S.cerevisiae core oligosaccarides have terminalα-1,3-glycan linkages which may make proteins hyper-antigenic and unsuitable for therapeutic use.The methylotrophic yeast,Pichia pastoris,is a single-cell eukaryote microorganism and has been successfully used to produce various recombinant heterologous proteins for both basic laboratory research and industrial manufacture. The advantages for P.pastoris to express heterologous proteins are following:it is easy to genetically manipulate and culture and can be grown to high cell densities;it can perform higher eukaryotic post-translational modifications,such as glycosylation and disulphide bond formation for the produce of soluble,correctly folded recombinant proteins;it has strong promoters which can drive the expression of interest protein. P.pastoris has been studied for protein-surface display based on the anchor system from S.cerevisiae.Tanino et al.immobilized the lipase from Rhizopus oryzae with a pro-sequence on the P.pastoris cell surface using the flocculation functional domain of Flolp(FS)from S.cerevisiae as an anchor.This system was noncovalently linked to the cell wall and suitable for the enzymes possessing activities at the C terminus.It is unsuitable for the enzymes that possess activities at the N terminus, which maybe limit the application of P.pastoris as a prospective cellular host for protein-surface display.This article is divided into three parts.PartⅠ:Construction of a novel Pichia pastoris cell-surface display system based on the cell wall protein Pir1Pir proteins are the covalently linked cell wall proteins in S.cerevisiae,which attach directly to theβ-1,3-glucan of the cell wall.Pir proteins do not contain a C-terminal GPI sequence and they are all processed by Kex2 protease.They can be released from intact cells by very mild alkaline treatment(30 mM NaOH,12 h,4℃). All Pir proteins have one or several units of an internal repetitive sequence at the N terminus.The target proteins can be fused to Pir proteins either in the middle or at the N- or C-terminus of Pir proteins.Pir proteins have been efficient anchors for the display of target proteins on the surface of S.cerevisiae.A novel system based on Pir1 from S.cerevisiae was developed for cell-surface display of heterologous proteins in P.pastoris with the alpha-factor secretion signal sequence.As a model protein,enhanced green fluorescence protein(EGFP)was fused to the N-terminal of the mature peptide of Pir1(Pir1-a).The expression of fusion protein EGFP-Pir1-a was irregular throughout the P.pastoris cell surface per detection by confocal laser scanning microscopy.A truncated sequence containing only the internal repetitive sequences of Pir1-a(Pir1-b)was used as a new anchor protein in further study.The fusion protein EGFP-Pir1-b was expressed uniformly on the cell surface.The fluorescence intensity of the whole yeast was measured by spectrofluorometer.Western blot confirmed that the fusion proteins were released from cell walls after mild alkaline treatment.The results indicate that a Pir1-based system can express proteins on the surface of P.pastoris and that the fusion proteins do not affect the manner in which Pir1 attaches to the cell wall.The repetitive sequences of Pir1 are required for cell wall retention,and the C-terminal sequence contributes to the irregular distribution of fusion proteins in P.pastoris.PartⅡ:Construction of a novel system for cell surface display of heterologous proteins on Pichia pastorisα-agglutinin belongs to another covalently linked cell wall proteins in S. cerevisiae,which have a GPI anchor at their C terminus and are attached toβ-1,3-glucan of the cell wall via a shortβ-1,6-glucan.GPI proteins can be released byβ-1,3- orβ-1,6-glucanase from cell wall.The C-terminal half ofα-agglutinin has been widely used to anchor heterologous proteins on the surface of S.cerevisiae.A versatile vector was developed for heterologous proteins display on the cell surface of P.pastoris using the C-terminal half ofα-agglutinin from S.cerevisiae as a membrane anchor under the control of the alcohol oxidase 1 promoter(pAOX1). Multiple cloning sites and the sequence encoding the Xpress epitope (-Asp-Leu-Tyr-Asp-Asp-Asp-Asp-Lys-)were introduced into the vector for insertion of heterologous genes and selective cleavage of target proteins.EGFP was used as a model protein to check the function of this vector.The expression of EGFP on the P. pastoris surface was confirmed by confocal laser scanning microscopy.Fluorescence microscopy and western blot analysis confirmed that EGFP can be successfully cleaved from the cell surface by treating with enterokinase.This system can be applied to purify heterologous proteins expressed in P.pastoris.PartⅢ:Comparison of cell wall proteins of Pir1 and the C-terminal half ofα-agglutinin as anchors for heterologous protein cell-surface displayThe mature peptide of Pir1 and the C-terminal half ofα-agglutinin have been proved capable of immobilizing EGFP on the cell surface of P.pastoris.The fraction of the total amount of fusion protein localized to the cell wall varied depending on both the anchor domain used and the fermentation conditions.The expression of fusion protein under various fermentation conditions,e.g.,the induction temperature, initial pH and methanol concentration were studied.And the ability of the mature peptide of Pir1 and the C-terminal half ofα-agglutinin to anchor EGFP to the cell wall of P.pastoris was compared under each of the optimal induction condition.It was found the constructed P.pastoris strain could display more fusion protein when the C-terminal half ofα-agglutinin was used as an anchor.