Abstract
A versatile vector was developed for heterologous proteins display on the cell surface of Pichia pastoris using the C-terminal half of α-agglutinin from Saccharomyces 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. Enhanced green fluorescence protein (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.
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References
Breinig F, Schmitt MJ (2002) Spacer-elongated cell wall fusion proteins improve cell surface expression in the yeast Saccharomyces cerevisiae. Appl Microbiol Biotechnol 58:637–644
Choi BK, Bobrowicz P, Davidson RC, Hamilton SR, Kung DH, Li H, Miele RG, Nett JH, Wildt S, Gerngross TU (2003) Use of combinatorial genetic libraries to humanize N-linked glycosylation in the yeast Pichia pastoris. Proc Natl Acad Sci USA 100:5022–5027
Gemmill TR, Trimble RB (1999) Overview of N- and O-linked oligosaccharide structures found in various yeast species. Biochim Biophys Acta 1426:227–237
Kato M, Maeda H, Kawakami M, Shiraga S, Ueda M (2005) Construction of a selective cleavage system for a protein displayed on the cell surface of yeast. Appl Microbiol Biotechnol 69:423–427
Kondo A, Ueda M (2004) Yeast cell-surface display—applications of molecular display. Appl Microbiol Biotechnol 64:28–40
Kusser KL, Randall TD (2003) Simultaneous detection of EGFP and cell surface markers by fluorescence microscopy in lymphoid tissues. J Histochem Cytochem 51:5–14
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685
Mergler M, Wolf K, Zimmermann M (2004) Development of a bisphenol A-adsorbing yeast by surface display of the Kluyveromyces yellow enzyme on Pichia pastoris. Appl Microbiol Biotechnol 63:418–421
Scott JK, Smith GP (1990) Searching for peptide ligands with an epitope library. Science 249:386–390
Stahl S, Uhlen M (1997) Bacterial surface display: trends and progress. Trends Biotechnol 15:185–192
Steidler L, Viaene J, Fiers W, Remaut E (1998) Functional display of a heterologous protein on the surface of Lactococcus lactis by means of the cell wall anchor of Staphylococcus aureus protein A. Appl Environ Microbiol 64:342–345
Tanino T, Fukuda H, Kondo A (2006) Construction of a Pichia pastoris cell-surface display system using Flo1p anchor system. Biotechnol Prog 22:989–993
Washida M, Takahashi S, Ueda M, Tanaka A (2001) Spacer-mediated display of active lipase on the yeast cell surface. Appl Microbiol Biotechnol 56:681–686
Acknowledgment
The project was supported by a key fund from Chinese Education Ministry (2005) and a Grant from National Natural Science Foundation of China (No. 30470399).
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Qingjie Wang and Lei Li contributed equally to this work.
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Wang, Q., Li, L., Chen, M. et al. Construction of a novel system for cell surface display of heterologous proteins on Pichia pastoris . Biotechnol Lett 29, 1561–1566 (2007). https://doi.org/10.1007/s10529-007-9430-6
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DOI: https://doi.org/10.1007/s10529-007-9430-6
Keywords
- α-Agglutinin
- Enhanced green fluorescence protein
- Pichia pastoris
- Surface display