Secretion and proteolysis of heterologous proteins fused to the Escherichia coli maltose binding protein in Pichia pastoris.
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.
Department of Chemistry, University of the Pacific, Stockton, CA 95211, USA.
Zhiguo
Z
Li
Wilson
W
Leung
Amy
A
Yon
John
J
Nguyen
Vincent C
VC
Perez
Jane
J
Vu
William
W
Giang
Linda T
LT
Luong
Tracy
T
Phan
Kate A
KA
Salazar
Seth R
SR
Gomez
Colin
C
Au
Fan
F
Xiang
David W
DW
Thomas
Andreas H
AH
Franz
Joan
J
Lin-Cereghino
Geoff P
GP
Lin-Cereghino
D000595:Amino Acid Sequence; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D058619:Maltose-Binding Proteins; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D033902:Periplasmic Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism
D029968:Escherichia coli Proteins; D058619:Maltose-Binding Proteins; D033902:Periplasmic Binding Proteins; D011993:Recombinant Fusion Proteins