Surface display of active lipase in Pichia pastoris using Sed1 as an anchor protein

Su, Guodong; Zhang, Xi; Lin, Ying

doi:10.1007/s10529-010-0270-4

Cited by 48 publications

(31 citation statements)

References 15 publications

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“…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.…”

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confidence: 99%

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

Zhang

Liang

Zhou

et al. 2013

Appl Environ Microbiol

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Glycosylphosphatidylinositol (GPI)-anchored glycoproteins have various intrinsic functions in yeasts and different uses in vitro.In the present study, the genome of Pichia pastoris GS115 was screened for potential GPI-modified cell wall proteins. Fifty putative GPI-anchored proteins were selected on the basis of (i) the presence of a C-terminal GPI attachment signal sequence, (ii) the presence of an N-terminal signal sequence for secretion, and (iii) the absence of transmembrane domains in mature protein. The predicted GPI-anchored proteins were fused to an alpha-factor secretion signal as a substitute for their own N-terminal signal peptides and tagged with the chimeric reporters FLAG tag and mature Candida antarctica lipase B (CALB). The expression of fusion proteins on the cell surface of P. pastoris GS115 was determined by whole-cell flow cytometry and immunoblotting analysis of the cell wall extracts obtained by ␤-1,3-glucanase digestion. CALB displayed on the cell surface of P. pastoris GS115 with the predicted GPI-anchored proteins was examined on the basis of potential hydrolysis of p-nitrophenyl butyrate. Finally, 13 proteins were confirmed to be GPI-modified cell wall proteins in P. pastoris GS115, which can be used to display heterologous proteins on the yeast cell surface.

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confidence: 99%

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

Zhang

Liang

Zhou

et al. 2013

Appl Environ Microbiol

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“…A few other studies have reported that cell surface display of enzymes can improve thermostability compared with purified enzymes. For example, Sed1 and Cwp2 are utilized as the anchor proteins to fuse lipase for surface display in Pichia pastoris and Saccharomyces cerevisiae, respectively [19,27].…”

Section: Discussionmentioning

confidence: 99%

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

Xue

Ding

2015

Curr Microbiol

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The cell surface display technique allows expression of target proteins or peptides on microbial cell surface by fusing an appropriate protein as an anchoring motif. Herein, we constructed an Escherichia coli-based whole-cell biocatalyst displaying Thermomyces lanuginosus DSM 5826 xylanase (XynA) on the cell surface and endowed the E. coli cells with the ability to degrade xylan. The XynA was fused in frame to the C-terminus of Lpp-OmpA fusion previously shown to direct various other heterologous proteins to E. coli cell surface. The expressed Lpp-OmpA-XynA fusion protein has a molecular weight of approximately 37 kDa, which was confirmed by SDS-PAGE and Western blot analysis. The enzyme activity of the surface-displayed xylanase showed clear halo around the colony. The XynA-displaying E. coli-based whole-cell biocatalyst xylanase activity was mainly detected with whole cells by determination of activity. The XynA-displaying E. coli-based whole-cell biocatalyst showed highest XynA activity at pH 6.2 and 65 °C, respectively. These results suggest that E. coli, which displayed the xylanase on its surface, could be used as a whole-cell biocatalyst in xylooligosaccharide production.

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“…Therefore, it is also a suitable host for use in large-scale fermentation cultures for molecular display technology. Both N-and C-terminusfree display systems of P. pastoris have been established using cell wall proteins -agglutinin, a-agglutinin, Flo1p, Sed1p, and Tip1p from S. cerevisiae, as described above Su et al, 2010;Tanino et al, 2006 .…”

Section: Molecular Display Technology Using Pichia Pastorismentioning

confidence: 99%

Bioadsorption Strategies with Yeast Molecular Display Technology

Shibasaki

Ueda

2014

Biocontrol Sci.

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Molecular display techniques using microbial cell surfaces have been widely developed in the past twenty years, and are useful tools as whole cell catalysts for various applications such as bioconversion, bioremediation, biosensing, and the screening system of protein libraries. Furthermore, different types of microbial cells among eukaryotic and prokaryotic strains have been investigated for their use in surface display technologies. Recently, several kinds of protein-displaying yeasts have been utilized as bioadsorbents in this platform technology. In particular, these trials have successfully expanded the possibility of applications to metal binding, affinity purification, and receptor-ligand interaction by using the yeast cell surface. In this mini review, we describe the general principles of molecular display technology using yeast cells and its applications, with a particular focus on bioadsorption.

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Surface display of active lipase in Pichia pastoris using Sed1 as an anchor protein

Cited by 48 publications

References 15 publications

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

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

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

Bioadsorption Strategies with Yeast Molecular Display Technology

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