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

Vectors and strains for expression.

Selection of both an appropriate expression vector and corresponding strain is crucial for successful expression of heterologous proteins in Pichia pastoris. This chapter explores both the standard and new vector/strain options available for protein expression in this yeast. Incorporated into expression vectors are selectable markers based on biosynthetic pathway genes, dominant drug resistance, or the P. pastoris formaldehyde dehydrogenase gene (FLD1). Novel strains available for expression include those that increase secretion of heterologous protein by overexpressing eukaryotic protein disulfide isomerase, and those that decrease hyperglycosylation or provide human-type glycosylation. This chapter also discusses methods to create multicopy strains that will potentially provide optimized expression of recombinant proteins in P. pastoris

Development of the G418 Resistance Gene as a Primary Selectable Marker for Pichia pastoris

There are relatively few dominant selectable markers available for the transformation of Pichia pastoris. The major markers, the zeocin and blasticidin resistance genes, require expensive antibiotics and extensive screening in order to isolate transformants with high copy number. The G418 resistance gene has been utilized for selection of multicopy strains, but only as a secondary selectable marker after primary selection with a biosynthetic marker such as HIS4. We have modified the G418 resistance gene so that it can now be used in P. pastoris for direct selection. Transformation with this new marker generates colonies of varying size on plates containing the antibiotic. Compared to small colonies, larger colonies harbor a greater number of plasmids containing the G418 resistance gene and express higher levels of a reporter gene that is carried in the vector. Besides adding greater flexibility to the P. pastoris system, this G418 selectable marker is more economical and provides an easier way to identify multicopy strains

Development of the G418 Resistance Gene as a Primary Selectable Marker for Pichia pastoris

There are relatively few dominant selectable markers available for the transformation of Pichia pastoris. The major markers, the zeocin and blasticidin resistance genes, require expensive antibiotics and extensive screening in order to isolate transformants with high copy number. The G418 resistance gene has been utilized for selection of multicopy strains, but only as a secondary selectable marker after primary selection with a biosynthetic marker such as HIS4. We have modified the G418 resistance gene so that it can now be used in P. pastoris for direct selection. Transformation with this new marker generates colonies of varying size on plates containing the antibiotic. Compared to small colonies, larger colonies harbor a greater number of plasmids containing the G418 resistance gene and express higher levels of a reporter gene that is carried in the vector. Besides adding greater flexibility to the P. pastoris system, this G418 selectable marker is more economical and provides an easier way to identify multicopy strains

Production of recombinant proteins in fermenter cultures of the yeast Pichia pastoris

The Pichia pastoris expression system offers economy, ease of manipulation, the ability to perform complex post-translational modifications, and high expression levels. Using this system, recent advances have been made in the quality of recombinant proteins in fermenter culture and in the quality of the protein product, namely improved secretion signals and glycosylation patterns

Expression of Foreign Genes in the yeast Pichia pastoris

Since 1985 the methylotrophic yeast Pichia pastoris has been successfully used for the production of over 300 recombinant proteins (1). (A brief description of published proteins with links to publications via PubMed is available at: http://public.kgi.edu/~cregg/Pichia.html). The increasing popularity of P. pastoris is attributed to: (a) the ease with which it can be genetically manipulated and cultured on a large scale; (b) its ability to produce foreign proteins either intracellularly or extracellularly at high levels; (c) its capability of performing many eukaryotic post-translational modifications, such as proteolytic processing, folding, disulfide bond formation, and glycosylation; and (d) its ready availability as a kit from Invitrogen Corporation (Carlsbad, CA). This article provides a general outline on the use of the P. pastoris expression system, summarizes the advantages and disadvantages of the system, and describes the characteristics of several new P. pastoris promoters, which may broaden the usefulness of the system. More detailed information on the P. pastoris expression system may be obtained in one of the many reviews on the system (2 and references therein) and from Invitrogen (http://www.invitrogen.com)

Preparation of the Yeast Pichia pastoris for Transmission Electron Microscopy.

The methylotropic yeast Pichia pastoris is a model organism for the study of autophagy and peroxisome biogenesis. Being able to look at the organism via transmission electron microscopy (TEM) can yield valuable data on the morphology of the secretory pathway and many other organelles of interest. However, preparing the yeast for TEM work can be very arduous and costly. One of the reasons P. pastoris is so hard to prepare for visualization is because its cell wall is very thick and tough compared to the membrane of a mammalian cell. Thus, P. pastoris is notoriously difficult to infiltrate with fixatives, a step necessary to maintain its ultrastructure. This article outlines an efficient and cost effective way to prepare P. pastoris for TEM without the need for certain specialized equipment. With this protocol, excellent pictures can be obtained by using the buffers, KMnO4, sorbitol, and PIPES, along with glutaraldehyde. These components preserve the ultrastructure of the yeast without any apparent artifactual change in morphology

Synthetic Pichia pastoris promoters based on AOX1 regulatory elements

During the last decade, the methylotrophic yeast, Pichia pastoris, has become a major eukaryotic host for recombinant protein production in both academic and industrial research. Up to now, the expression of more than 500 proteins has been reported. One major reason for the success of this yeast as an expression system is the inducible promoter of its alcohol oxidase I (AOX1) gene. Its key features include an exceptional expression strength as well as a very strong glucose repression. By computational sequence analysis several putative cis-acting elements could be identified within the AOX1 promoter sequence. Based on this sequence analyses, we performed deletion studies and identified both, positively and negatively acting promoter elements. Consequently, these elements were tested by adding them to basal promoter elements and finally they were rearranged to generate synthetic and hybrid promoter libraries with different expression levels and regulation patterns