Ilya Tolstorukov

Expression in the yeast Pichia pastoris.

The yeast Pichia pastoris has become the premier example of yeast species used for the production of recombinant proteins. Advantages of this yeast for expression include tightly regulated and efficient promoters and a strong tendency for respiratory growth as opposed to fermentative growth. This chapter assumes the reader is proficient in molecular biology and details the more yeast specific procedures involved in utilizing the P. pastoris system for gene expression. Procedures to be found here include: strain construction by classical yeast genetics, the logic in selection of a vector and strain, preparation of electrocompetent yeast cells and transformation by electroporation, and the yeast colony western blot or Yeastern blot method for visualizing secreted proteins around yeast colonies.

Mxr1p, a Key Regulator of the Methanol Utilization Pathway and Peroxisomal Genes in Pichia pastoris

ABSTRACT Growth of the yeast Pichia pastoris on methanol induces the expression of genes whose products are required for its metabolism. Three of the methanol pathway enzymes are located in an organelle called the peroxisome. As a result, both methanol pathway enzymes and proteins involved in peroxisome biogenesis (PEX proteins) are induced in response to this substrate. The most highly regulated of these genes is AOX1, which encodes alcohol oxidase, the first enzyme of the methanol pathway, and a peroxisomal enzyme. To elucidate the molecular mechanisms responsible for methanol regulation, we identify genes required for the expression of AOX1. Mutations in one gene, named MXR1 (methanol expression regulator 1), result in strains that are unable to (i) grow on the peroxisomal substrates methanol and oleic acid, (ii) induce the transcription of AOX1 and other methanol pathway and PEX genes, and (iii) form normal-appearing peroxisomes in response to methanol. MXR1 encodes a large protein with a zinc finger DNA-binding domain near its N terminus that has similarity to Saccharomyces cerevisiae Adr1p. In addition, Mxr1p is localized to the nucleus in cells grown on methanol or other gluconeogenic substrates. Finally, Mxr1p specifically binds to sequences upstream of AOX1. We conclude that Mxr1p is a transcription factor that is necessary for the activation of many genes in response to methanol. We propose that MXR1 is the P. pastoris homologue of S. cerevisiae ADR1 but that it has gained new functions and lost others through evolution as a result of changes in the spectrum of genes that it controls.

Genetic control of cell type and complex organization of the mating type locus in the yeast Pichia pinus

SummaryGenetic mechanisms of switching the mating type locus MAT1 in the homothallic yeast Pichia pinus were studied. By analysis of mutations affecting MAT1 complex structural and functional organization of this locus was shown. The existence of two functional regions in MAT1 is postulated. Region I controls mating ability of haploid cells, determines the neutrality of heterozygous cells and regulates the work of Region II. Region II controls meiosis and/or sporulation in the cultures heterozygous for Region I as well as controls switching MAT1 ⇋ MAT1α in haploid cells.

A scalable low-cost cGMP process for clinical grade production of the HIV inhibitor 5P12-RANTES in Pichia pastoris

In the continued absence of an effective anti-HIV vaccine, approximately 2 million new HIV infections occur every year, with over 95% of these in developing countries. Calls have been made for the development of anti-HIV drugs that can be formulated for topical use to prevent HIV transmission during sexual intercourse. Because these drugs are principally destined for use in low-resource regions, achieving production costs that are as low as possible is an absolute requirement. 5P12-RANTES, an analog of the human chemokine protein RANTES/CCL5, is a highly potent HIV entry inhibitor which acts by achieving potent blockade of the principal HIV coreceptor, CCR5. Here we describe the development and optimization of a scalable low-cost production process for 5P12-RANTES based on expression in Pichia pastoris. At pilot (150 L) scale, this cGMP compliant process yielded 30 g of clinical grade 5P12-RANTES. As well as providing sufficient material for the first stage of clinical development, this process represents an important step towards achieving production of 5P12-RANTES at a cost and scale appropriate to meet needs for topical HIV prevention worldwide.

Pilot study of a rapid and minimally instrumented sputum sample preparation method for molecular diagnosis of tuberculosis.

Nucleic acid amplification testing (NAAT) enables rapid and sensitive diagnosis of tuberculosis (TB), which facilitates treatment and mitigates transmission. Nucleic acid extraction from sputum constitutes the greatest technical challenge in TB NAAT for near-patient settings. This report presents preliminary data for a semi-automated sample processing method, wherein sputum is disinfected and liquefied, followed by PureLyse® mechanical lysis and solid-phase nucleic acid extraction in a miniaturized, battery-operated bead blender. Sputum liquefaction and disinfection enabled a >104 fold reduction in viable load of cultured Mycobacterium tuberculosis (M.tb) spiked into human sputum, which mitigates biohazard concerns. Sample preparation via the PureLyse® method and a clinically validated manual method enabled positive PCR-based detection for sputum spiked with 104 and 105 colony forming units (cfu)/mL M.tb. At 103 cfu/mL sputum, four of six and two of six samples amplified using the comparator and PureLyse® method, respectively. For clinical specimens from TB cases and controls, the two methods provided 100% concordant results for samples with 1 mL input volume (N = 41). The semi-automated PureLyse® method therefore performed similarly to a validated manual comparator method, but is faster, minimally instrumented, and can be integrated into TB molecular diagnostic platforms designed for near-patient low-resource settings.

Expression of Recombinant Genes in the Yeast Pichia pastoris

The synthesis of specific recombinant proteins using single‐celled organisms from bacteria to mammalian tissue culture cells has become a major source of biopharmaceutical products for the industry and a source of a wide variety of proteins for academic research. A range of organisms are utilized for this purpose. One of the newest and most promising of these is the yeast Pichia pastoris. This article provides detailed basic protocols for the expression of heterologous genes and the synthesis of recombinant proteins utilizing this yeast. Specifically provided are protocols for the insertion of foreign vector DNAs into the yeast by electroporation, amplification of vector sequences by the post‐translational vector amplification (PTVA) method, and growth and expression of foreign genes in shake flask cultures. Curr. Protoc. Essential Lab. Tech. 4:13.2.1‐13.2.14.

Electroporation of Pichia pastoris

The introduction of foreign DNA into the yeast Pichia pastoris is similar to the processes used with Saccharomyces cerevisiae and routinely performed by the electroporation of plasmid DNA into electro-competent (E-comp) P. pastoris cells (Becker and Guarente, Methods Enzymol 194:182–187, 1991). Transformation of spheroplasts is more difficult (Cregg et al., Mol Cell Biol 5:3376–3385, 1985), and current methods to make chemically competent P. pastoris cells results in significantly fewer transformants (Pichia protocols, 2nd edn, Totowa, 27–42). Following are protocols to prepare E-comp P. pastoris cells, and to electroporate plasmid DNA into these cells to generate transformed cells.