Surface display of human lactoferrin using a glycosylphosphatidylinositol-anchored protein of Saccharomyces cerevisiae in Pichia pastoris.
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.
Department of Bioscience and Biotechnology, College of Natural Sciences, Hankuk University of Foreign Studies, Yongin, Gyeonggi, 449-791, Korea.
Jae-Hyung
JH
Jo
Eun-Mi
EM
Im
Seung-Hwan
SH
Kim
Hyune-Hwan
HH
Lee
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D019677:Artificial Gene Fusion; D001483:Base Sequence; D001709:Biotechnology; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D058851:GPI-Linked Proteins / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D005822:Genetic Vectors; D006023:Glycoproteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007781:Lactoferrin / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013211:Staphylococcus aureus / Q000187:drug effects
D000900:Anti-Bacterial Agents; D002352:Carrier Proteins; D017931:DNA Primers; D058851:GPI-Linked Proteins; D006023:Glycoproteins; C490098:LTF protein, human; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C070948:TIP1 protein, S cerevisiae; D007781:Lactoferrin