Overcoming Obstacles in Protein Expression in the Yeast Pichia pastoris: Interviews of Leaders in the Pichia Field

The yeast Pichia pastoris (also known as Komagataella pastoris) has been used for over 30 years to produce thousands of valuable, heterologous proteins, such as insulin to treat diabetes and antibodies to prevent migraine headaches. Despite its success, there are some common, stubborn problems encountered by research scientists when they try to use the yeast to produce their recombinant proteins. In order to provide those working in this field with strategies to overcome these common obstacles, nine experts in P. pastoris protein expression field were interviewed to create a written review and video (https://www.youtube.com/watch?v=Q1oD6k8CdG8). This review describes how each respected scientist addressed a specific challenge, such as identifying high expression strains, improving secretion efficiency and decreasing hyperglycosylation. Their perspective and practical advice can be a tool to help empower others to express challenging proteins in this popular recombinant host

Robert Matijašić, Povijest hrvatskih zemalja u kasnoj antici od Dioklecijana do Justinijana

Introduction Pichia pastoris is a methylotrophic yeast that has been genetically engineered to express heterologous. In recent 20 years, over 700 proteins from bacteria to humans have been produced in this yeast. MBP (maltose binding protein) has been utilized as a translational fusion partner to improve the expression of foreign proteins made in E. coli. We initially explored whether MBP would serve as an expression enhancer and purification tag in Pichia pastoris, a popular eukaryotic host for heterologous protein expression. Methods SDS-PAGE and Western analysis were applied to analyze the protein expression. The secreted fusion proteins were purified by the amylose resin, digested by trypsin or endoproteinase Asp-N, and subjected to mass spectrometric analysis. Preliminary results When MBP was fused as an N-terminal partner to several cargo proteins (the two proteins were separated by a Factor Xa protease site) expressed in this yeast, proteolysis occurred between the two peptides and only MBP reached the extracellular region, which suggested that the fusion protein had been proteolyzed between MBP and cargo proteins. Furthermore, western analysis indicated the fusion proteins had been cleaved inside the yeast. Mass spectrometry analysis of MBP-FXa-FKBP12 demonstrated the Cterminus of that fusion protein was IEGR, the FXa sequence. Extensive mutagenesis of this spacer region between MBP and FKBP12 could not inhibit the cleavage. Mass spectrometric data indicated different C-termini in these mutant proteins, suggesting that different cleavage sites were used in the MBP fusions. These results provide new insights into the role of proteases in this expression system

Endosome to Golgi Retrieval of the Vacuolar Protein Sorting Receptor, Vps10p, Requires the Function of the VPS29, VPS30, and VPS35 Gene Products

Mutations in the S. cerevisiae VPS29 and VPS30 genes lead to a selective protein sorting defect in which the vacuolar protein carboxypeptidase Y (CPY) is missorted and secreted from the cell, while other soluble vacuolar hydrolases like proteinase A (PrA) are delivered to the vacuole. This phenotype is similar to that seen in cells with mutations in the previously characterized VPS10 and VPS35 genes. Vps10p is a late Golgi transmembrane protein that acts as the sorting receptor for soluble vacuolar hydrolases like CPY and PrA, while Vps35p is a peripheral membrane protein which cofractionates with membranes enriched in Vps10p. The sequences of the VPS29, VPS30, and VPS35 genes do not yet give any clues to the functions of their products. Each is predicted to encode a hydrophilic protein with homologues in the human and C. elegans genomes. Interestingly, mutations in the VPS29, VPS30, or VPS35 genes change the subcellular distribution of the Vps10 protein, resulting in a shift of Vps10p from the Golgi to the vacuolar membrane. The route that Vps10p takes to reach the vacuole in a vps35 mutant does not depend upon Sec1p mediated arrival at the plasma membrane but does require the activity of the pre-vacuolar endosomal t-SNARE, Pep12p. A temperature conditional allele of the VPS35 gene was generated and has been found to cause missorting/secretion of CPY and also Vps10p to mislocalize to a vacuolar membrane fraction at the nonpermissive temperature. Vps35p continues to cofractionate with Vps10p in vps29 mutants, suggesting that Vps10p and Vps35p may directly interact. Together, the data indicate that the VPS29, VPS30, and VPS35 gene products are required for the normal recycling of Vps10p from the prevacuolar endosome back to the Golgi where it can initiate additional rounds of vacuolar hydrolase sorting

Characterization of the role of BGS13 in the secretory mechanism of Pichia pastoris

The methylotrophic yeast Pichia pastoris has been utilized for heterologous protein expression for over 30 years. Because P. pastoris secretes few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, some recombinant proteins intended for secretion are retained within the cell. A mutant strain isolated in our laboratory, containing a disruption of the BGS13 gene, displayed elevated levels of secretion for a variety of reporter proteins. The Bgs13 peptide (Bgs13p) is similar to the Saccharomyces cerevisiae protein kinase C 1 protein (Pkc1p), but its specific mode of action is currently unclear. To illuminate differences in the secretion mechanism between the wild-type (wt) strain and the bgs13 strain, we determined that the disrupted bgs13 gene expressed a truncated protein that had reduced protein kinase C activity and a different location in the cell, compared to the wt protein. Because the Pkc1p of baker’s yeast plays a significant role in cell wall integrity, we investigated the sensitivity of the mutant strain’s cell wall to growth antagonists and extraction by dithiothreitol, determining that the bgs13 strain cell wall suffered from inherent structural problems although its porosity was normal. A proteomic investigation of the bgs13 strain secretome and cell wall-extracted peptides demonstrated that, compared to its wt parent, the bgs13 strain also displayed increased release of an array of normally secreted, endogenous proteins, as well as endoplasmic reticulum-resident chaperone proteins, suggesting that Bgs13p helps regulate the unfolded protein response and protein sorting on a global scale. IMPORTANCE The yeast Pichia pastoris is used as a host system for the expression of recombinant proteins. Many of these products, including antibodies, vaccine antigens, and therapeutic proteins such as insulin, are currently on the market or in late stages of development. However, one major weakness is that sometimes these proteins are not secreted from the yeast cell efficiently, which impedes and raises the cost of purification of these vital proteins. Our laboratory has isolated a mutant strain of Pichia pastoris that shows enhanced secretion of many proteins. The mutant produces a modified version of Bgs13p. Our goal is to understand how the change in the Bgs13p function leads to improved secretion. Once the Bgs13p mechanism is illuminated, we should be able to apply this understanding to engineer new P. pastoris strains that efficiently produce and secrete life-saving recombinant proteins, providing medical and economic benefits

Positive selection of novel peroxisome biogenesis-defective mutants of the yeast Pichia pastoris

We have developed two novel schemes for the direct selection of peroxisome-biogenesis-defective (pex) mutants of the methylotrophic yeast Pichia pastoris. Both schemes take advantage of our observation that methanol-induced pex mutants contain little or no alcohol oxidase (AOX) activity. AOX is a peroxisomal matrix enzyme that catalyzes the first step in the methanol-utilization pathway. One scheme utilizes allyl alcohol, a compound that is not toxic to cells but is oxidized by AOX to acrolein, a compound that is toxic. Exposure of mutagenized populations of AOX-induced cells to allyl alcohol selectively kills AOX-containing cells. However, pex mutants without AOX are able to grow. The second scheme utilizes a P. pastoris strain that is defective in formaldehyde dehydrogenase (FLD), a methanol pathway enzyme required to metabolize formaldehyde, the product of AOX. AOX-induced cells of fld1 strains are sensitive to methanol because of the accumulation of formaldehyde. However, fld1 pex mutants, with little active AOX, do not efficiently oxidize methanol to formaldehyde and therefore are not sensitive to methanol. Using these selections, new pex mutant alleles in previously identified PEX genes have been isolated along with mutants in three previously unidentified PEX group

The Power of Pichia

Heterologous protein expression consists of engineering an organism to express a protein which it does not produce in nature, such as human insulin made by a microbe. The methylotrophic yeast Pichia pastoris has been a popular host for heterologous protein expression in order to produce thousands of recombinant protein products for research, clinical, and industrial purposes. Because this yeast secretes very few of its own proteins, the exported recombinant protein is the major polypeptide in the extracellular medium, making purification relatively easy. Unfortunately, a disadvantage to programmed export is that some recombinant proteins intended for secretion are retained within in the cell and may be subsequently degraded. A mutant strain isolated in our lab, containing a disruption of the BGS13 gene, has displayed elevated levels of secretion for a variety of reported proteins. Therefore, we are trying to understand how mutation of this BGS13 gene leads to an abnormal Bgs13 protein which in turn results in improved secretion of many different recombinant proteins. Our long term goal is to apply this understanding of the BGS13 gene to create new versions of Pichia pastoris expression strains that will be able to secrete larger amounts of valuable recombinant proteins