Surface display of human lactoferrin using a glycosylphosphatidylinositol-anchored protein of Saccharomyces cerevisiae in Pichia pastoris

Abstract

A cell surface display system was developed in Pichia pastoris using the gene TIP1, encoding the glycosylphosphatidylinositol (GPI)-anchored protein of Saccharomyces cerevisiae (ScTIP). Human lactoferrin cDNA (hLf) was fused to a full-length TIP1 DNA (ScTIP ( 630 )) or a short-TIP1 fragment (ScTIP ( 120 )) encoding the 40 C-terminal amino acids of ScTIP. Both hLf-ScTIP fusion genes were expressed in P. pastoris SMD 1168. The fused protein was detected by western blotting after extraction of the lysed recombinant cells with Triton X-100, urea, and Triton X-100 plus urea, suggesting that the hLf is associated with the membrane. The localization of surface-displayed hLf was confirmed by immunofluorescence confocal microscopy and flow cytometric analysis using FITC-labeled anti-hLf antibody, suggesting that hLf was successfully located at the surface of P. pastoris. The intact recombinant cells and cell lysates showed antibacterial activity against target microorganisms, meaning that the expressed hLf was biologically active. The results indicated that the ScTIP anchoring motif is useful for cell surface display of foreign proteins in P. pastoris.

Screening for Glycosylphosphatidylinositol-Modified Cell Wall Proteins in Pichia pastoris and Their Recombinant Expression on the Cell Surface

“…The disruption of GAS1 in P. pastoris production strains for obtaining human trypsinogen and human serum albumin did not result in an enhancement of product secretion, whereas Rhizopus oryzae lipase secretion could be improved 2-fold (17). In addition, GPI-modified cell wall proteins from S. cerevisiae, such as ␣-agglutinin, Tip1p, Flo1p, and Sed1p, have been used as anchor proteins for P. pastoris cell surface display of heterologous proteins (18)(19)(20). However, very few endogenous GPImodified cell wall proteins of P. pastoris have been confirmed and used in P. pastoris cell surface display.…”

Applications of Yeast Surface Display for Protein Engineering

“…lipase were used to enrich docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from fish oil 115 ; yeast displaying Corynebacterium diphtheria sialidase were used to transfer sialic acids for glycoprotein remodeling 116 , and yeast displaying glucose oxidase were used for electrochemical glucose sensing 117 . Yeast have also been functionalized with antimicrobial peptides 118,119 , pathogenic proteins for oral vaccine delivery 120–122 , and metal-binding proteins for bioadsporption of various metals 123–129 . These and other examples have recently been reviewed 69 .…”

Display of Fungi Xylanase on Escherichia coli Cell Surface and Use of the Enzyme in Xylan Biodegradation

“…In recent years, there has been considerable progress in the development of expression systems for the display of heterologous peptides and proteins on the surface of bacteriophages, bacteria, yeasts, fungi, and mammalian cells [10,12,18,24,31]. Microbial cell surface display has a wide range of biotechnological and industrial applications in areas such as whole-cell biocatalysis for bioconversion, biosensing, biosorption, and immobilization [17].…”