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Article
Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein
Protein Expression & Purification
  • Zhiguo Li, University of the Pacific
  • Wilson Leung, University of the Pacific
  • Amy Yon, University of the Pacific
  • John Nguyen, University of the Pacific
  • Vincent C. Perez, University of the Pacific
  • Jane Vu, University of the Pacific
  • William Giang, University of the Pacific
  • Linda T. Luong, University of the Pacific
  • Tracy Phan, University of the Pacific
  • Kate A. Salazar, University of the Pacific
  • Seth R. Gomez, University of the Pacific
  • Colin Au, University of the Pacific
  • Fan Xiang, Shimadzu Biotech Corporation
  • David W. Thomas, University of the Pacific
  • Andreas H. Franz, University of the Pacific
  • Joan Lin-Cereghino, University of the Pacific
  • Geoff P. Lin-Cereghino, University of the Pacific
Document Type
Article
Department
Chemistry
DOI
10.1016/j.pep.2010.03.004
Publication Date
7-1-2010
Disciplines
Abstract
The Escherichia coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. When located N-terminal to its cargo protein, MBP increases the solubility of intracellular proteins and improves the export of secreted proteins in bacterial systems. We initially explored whether MBP would have the same effect in the methylotrophic yeast Pichia pastoris, a popular eukaryotic host for heterologous protein expression. When MBP was fused as an N-terminal partner to several C-terminal cargo proteins expressed in this yeast, proteolysis occurred between the two peptides, and MBP reached the extracellular region unattached to its cargo. However, in two of three instances, the cargo protein reached the extracellular region as well, and its initial attachment to MBP enhanced its secretion from the cell. Extensive mutagenesis of the spacer region between MBP and its C-terminal cargo protein could not inhibit the cleavage although it did cause changes in the protease target sites in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that an uncharacterized P. pastoris protease attacked at different locations in the region C-terminal of the MBP domain, including the spacer and cargo regions, but the MBP domain could still act to enhance the secretion of certain cargo proteins.
Citation Information
Zhiguo Li, Wilson Leung, Amy Yon, John Nguyen, et al.. "Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein" Protein Expression & Purification Vol. 72 Iss. 1 (2010) p. 113 - 124 ISSN: 1046-5928
Available at: http://works.bepress.com/andreas-franz/48/