Surface display of active lipase in Pichia pastoris using Sed1 as an anchor protein.
A Pichia pastoris cell-surface display system was constructed using the Sed1 anchor system that has been developed in Saccharomyces cerevisiae. Candida antarctica lipase B (CALB) was used as the model protein and was fused to an anchor that consisted of 338 amino acids of Sed1. The resulting fusion protein CALBSed1 was expressed under the control of the alcohol oxidase 1 promoter (pAOX1). Immunofluorescence microscopy of immunolabeled Pichia pastoris revealed that CALB was displayed on the cell surface. Western blot analysis showed that the fusion protein CALBSed1 was attached covalently to the cell wall and was highly glycosylated. The hydrolytic activity of the displayed CALB was more than 220 U/g dry cells after 120 h of culture. The displayed protein also exhibited a higher degree of thermostability than free CALB.
Su
Guo-dong
GD
Guangdong Key Laboratory of Fermentation and Enzyme Engineering, School of Bioscience and Bioengineering, South China University of Technology, Guangzhou 510006, People's Republic of China.
Zhang
Xi
X
Lin
Ying
Y
D015153:Blotting, Western; D005455:Fluorescent Antibody Technique; D005656:Fungal Proteins; D008049:Lipase / Q000235:genetics / Q000378:metabolism*; D008562:Membrane Glycoproteins / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013696:Temperature
D005656:Fungal Proteins; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; C077394:SED1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D008049:Lipase; C431349:lipase B, Candida antarctica