Secretion and proteolysis of heterologous proteins fused to the E. coli maltose binding protein in Pichia pastoris
Document Type
Poster
Department
Biological Sciences
Conference Title
Pichia 2009 Conference
Location
Tucson, AZ
Conference Dates
October 18-21, 2009
Date of Presentation
10-18-2009
Abstract
The E. coli maltose binding protein (MBP) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. MBP has been shown to increase the solubility of intracellular proteins as well as improve the export of secreted proteins. We initially explored whether MBP would have the same effect in Pichia pastoris.. 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 only MBP reached the extracellular region. 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 site in the fusion proteins, as determined by mass spectrometry. Taken together, these results suggested that the three dimensional structure of MBP triggered attack by an uncharacterized P. pastoris. protease at a nonspecific region C-terminal of the MBP domain. However, MBP was able to serve as a secretion enhancer when it was fused as a C-terminal peptide to an N-terminal cargo protein. These studies provide new insights into the role of proteases and fusion partners in the secretory mechanism of P. pastoris.
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.,
Xiang, F.,
Franz, A. H.,
Lin-Cereghino, J.,
&
Lin-Cereghino, G. P.
(2009).
Secretion and proteolysis of heterologous proteins fused to the E. coli maltose binding protein in Pichia pastoris.
Paper presented at Pichia 2009 Conference in Tucson, AZ.
https://scholarlycommons.pacific.edu/cop-facpres/1244
