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

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.

Figure 1. Diagram of Representative Constructs Generated for Fusion Protein Studies

Constructs were made with combinations of six elements: the MATα prepro peptide (MATα), E.coli maltose binding protein (MBP), a protease site for Factor Xa (F) or enterokinase (E), a cargo protein (CARGO), and a myc tag (✦) for antibody detection.

Figure 2. Silver stain and western analysis of pJV1 (MBP-FXa) and pJV2 (MBP-FXa-FKBP12-myc-His6) expression

Strains containing the expression plasmids were grown in BMGY glycerol medium and then induced in BMMY methanol medium, as described in the Methods. Aliquots of culture supernatant, corresponding to equivalent numbers of cells (based on OD₆₀₀), were subjected to SDS-PAGE and then visualized with either (A) silver stain or (B) western blot. Westerns utilized a primary rabbit anti-MBP antibody followed by a secondary goat anti-rabbit antibody. Lane M: molecular weight marker; lane 1: MBP-2 standard; lane 2: pPICZαB supernatant; lane 3: pJV1 supernatant; lane 4: pJV2 supernatant.

Figure 3. Western analysis of pJV2 (MBP-FXa-FKBP12-myc-His6), pJV3 (MBP-FXa-Spe9-myc-His6) and pJV4 (MBP-FXa-EGFP-myc-His6) proteins with anti-MBP antibody

Lane 1: MBP-2 standard; lane 2: pPICZαB supernatant; lane3: empty; lane 4: pJV1 supernatant; lane 5: pJV2 supernatant; lane 6: pJV2 concentrated supernatant; lane 7: pJV3 supernatant; lane 8: pJV4 supernatant.

Figure 4. Western analysis of pJV2 (MBP-FXa-FKBP12-myc-His6) supernatant and cell extract with anti-MBP antibody

Volumes of culture supernatant, corresponding to equivalent numbers of cells (based on OD₆₀₀), were analyzed in supernatant lanes while equivalent masses of cell extract were examined in cell extract lanes. Lane 1: pJV2 cell extract; lane 2: pPICZαB cell extract; lane 3: pPICZαB supernatant; lane 4: pJV2 supernatant; lane 5: MBP-2 standard

Figure 5. MALDI-TOF analysis of trypsin digestion of pJV2 (MBP-FXa-FKBP12-myc-His6) product

Figure 6. (A) MALDI-TOF analysis of endoproteinase Asp-N digestion of pJV2 (MBP-FXa-FKBP12-myc-His6) product

Figure 7. MS/MS of selected ion ³⁶⁵DAQTNSSSNNNNNNNNNNLGIEGR³⁸⁸ in MALDI-QIT-TOF

Figure 8. Western of pJV2, pHL-IEGV, pHL-ΔIEGR and pHL-ΔpolyN with anti-MBP antibody

Lane 1: MBP-2 standard; lane 2: pJV2 supernatant; lane 3: pPICZαB supernatant; lane 4: pHL-IEGV supernatant; lane 5: pHL-ΔIEGR supernatant; lane 6: pHL-ΔpolyN supernatant.

Figure 9. SDS-PAGE with (A) silver staining and (B) western analysis with anti-MBP antibody of pAW1 (MBP-EK-myc-His6), pAW2 (MBP-EK-FKBP12-myc-His6) and pAW3 (MBP-EK-EGFP-myc-His6)

Lane M: Marker; lane 1: MBP-2; lane 2: pPICZαB supernatant; lane 3: empty; lane 4: pAW1 supernatant; lane 5: pAW2 supernatant; lane 6: pAW3 supernatant.

Figure 10. Western analysis of supernatants to determine fates of cargo proteins

Parts A and B. Lane 1:pCA2 (FKBP-myc-His6) supernatant and lane 2: pJV2 (MBP-FXa-FKBP12-myc-His6) supernatant probed with anti-MBP (A) and anti-FKBP12 (B) antibodies. Part C. Lane 1: MBP-2 standard; lane 2: pPICZαB supernatant; lane 3: pJV4 (MBP-FXa-EGFP-myc-His6) supernatant; lane 4: pKS1 (EGFP-myc-His6) supernatant probed with anti-MBP antibody. Part D. Lane 1: E.coli expressing pGLO plasmid (EGFP standard); lane 2: pPICZαB supernatant; lane 3: pJV4 (MBP-FXa-EGFP-myc-His6) supernatant; lane 4: pKS1 (EGFP-myc-His6) supernatant probed with anti-EGFP antibody. Part E. Lane 1: MBP-2 standard; lane 2: pCH1 (HRP) supernatant; lane 3: pHL2 (MBP-FXa-HRP-myc-His6) supernatant probed with anti-MBP antibody. Part F. Lane 1: native horseradish peroxidase; lane 2: pCH1 (HRP) supernatant; lane 3: pHL2 (MBP-FXa-HRP-myc-His6) supernatant probed with anti-HRP antibody.