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Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris
Protein Expression & Purification
  • Pachai S. Moua, University of the Pacific
  • Alfonso Gonzalez, University of the Pacific
  • Kristin T. Oshiro, University of the Pacific
  • Vivian Tam, University of the Pacific
  • Zhiguo Harry Li, University of the Pacific
  • Jennifer Chang, University of the Pacific
  • Wilson Leung, University of the Pacific
  • Amy Yon, University of the Pacific
  • Der Thor, University of the Pacific
  • Sri Venkatram, University of the Pacific
  • Andreas H. Franz, University of the Pacific
  • Douglas D. Risser, University of the Pacific
  • Joan Lin-Cereghino, University of the Pacific
  • Geoff P. Lin-Cereghino, University of the Pacific
Document Type
Publication Date
The Escherichia coli maltose binding protein (MBP) is an N-terminal fusion partner that was shown to enhance the secretion of some heterologous proteins from the yeast Pichia pastoris, a popular host for recombinant protein expression. The amount of increase in secretion was dependent on the identity of the cargo protein, and the fusions were proteolyzed prior to secretion, limiting its use as a purification tag. In order to overcome these obstacles, we used the MBP as C-terminal partner for several cargo peptides. While the Cargo-MBP proteins were no longer proteolyzed in between these two moieties when the MBP was in this relative position, the secretion efficiency of several fusions was lower than when MBP was located at the opposite end of the cargo protein (MBP-Cargo). Furthermore, fluorescence analysis suggested that the MBP-EGFP and EGFP-MBP proteins followed different routes within the cell. The effect of several Pichia pastoris beta-galactosidase supersecretion (bgs) strains, mutants showing enhanced secretion of select reporters, was also investigated on both MBP-EGFP and EGFP-MBP. While the secretion efficiency, proteolysis and localization of the MBP-EGFP was influenced by the modified function of Bgs13, EGFP-MBP behavior was not affected in the bgs strain. Taken together, these results indicate that the location of the MBP in a fusion affects the pathway and trans-acting factors regulating secretion in P. pastoris.
Citation Information
Pachai S. Moua, Alfonso Gonzalez, Kristin T. Oshiro, Vivian Tam, et al.. "Differential secretion pathways of proteins fused to the Escherichia coli maltose binding protein (MBP) in Pichia pastoris" Protein Expression & Purification Vol. 124 Iss. 1 (2016) p. 1 - 9 ISSN: 1046-5928
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