Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein

Authors

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 PacificFollow
Andreas H. Franz, University of the PacificFollow
Joan Lin-Cereghino, University of the PacificFollow
Geoff P. Lin-Cereghino, University of the PacificFollow

Document Type

Article

Publication Title

Protein Expression and Purification

Department

Chemistry

ISSN

1046-5928

Volume

72

Issue

1

DOI

10.1016/j.pep.2010.03.004

First Page

113

Last Page

124

Publication Date

7-1-2010

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.

Recommended Citation

Li, Z., Leung, W., Yon, A., Nguyen, J., Perez, V. C., Vu, J., Giang, W., Luong, L. T., Phan, T., Salazar, K. A., Gomez, S. R., Au, C., Xiang, F., Thomas, D. W., Franz, A. H., Lin-Cereghino, J., & Lin-Cereghino, G. P. (2010). Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein. Protein Expression and Purification, 72(1), 113–124. DOI: 10.1016/j.pep.2010.03.004
https://scholarlycommons.pacific.edu/cop-facarticles/114