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.
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"Technologies) and ligated as an in-frame fusion directly after the KREAEA sequence of the alpha mating factor encoded by the pJAZaMF expression vector  (Biogrammatics), which carries the Alcohol Oxidase 1 (AOX1) promoter, allowing M A N U S C R I P T A C C E P T E D "
[Show abstract][Hide abstract] ABSTRACT: 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.
Preview · Article · Oct 2015 · Protein Expression and Purification
"Alternatively, we recently established a recombinant expression approach for preparation of the relaxin family peptides through overexpression of designed single-chain precursors in E. coli and subsequent in vitro refolding and maturation (Luo et al. 2010a, b; Zhang et al. 2012a, b). In recent years, the Pichia expression system has been used for the successful preparation of some disulfide-rich proteins (Cregg et al. 2009; Damasceno et al. 2012; Li et al. 2007; Mattanovich et al. 2012). As the Pichia expression system can form correct disulfide linkages in vivo, in the present study, we used it for efficient preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression of a designed single-chain precursor and subsequent in vitro enzymatic maturation. "
[Show abstract][Hide abstract] ABSTRACT: Relaxin family peptides are a group of peptide hormones with divergent biological functions. Mature relaxin family peptides are typically composed of two polypeptide chains with three disulfide linkages, rendering their preparation a challenging task. In the present study, we established an efficient approach for preparation of the chimeric relaxin family peptide R3/I5 through secretory overexpression in Pichia pastoris and in vitro enzymatic maturation. A designed single-chain R3/I5 precursor containing the B-chain of human relaxin-3 and the A-chain of human INSL5 was overexpressed in PichiaPink strain 1 by high-density fermentation in a two-liter fermenter, and approximately 200 mg of purified precursor was obtained from one liter of the fermentation supernatant. We also developed an economical approach for preparation of the uniformly (15)N-labeled R3/I5 precursor by culturing in shaking flasks, and approximately 15 mg of purified (15)N-labeled precursor was obtained from one liter of the culture supernatant. After purification by cation ion-exchange chromatography and reverse-phase high performance liquid chromatography, the R3/I5 precursor was converted to the mature two-chain form by sequential treatment with endoproteinase Lys-C and carboxypeptidase B. The mature R3/I5 peptide had an α-helix-dominated conformation and retained full receptor-binding and receptor activation activities. Thus, Pichia overexpression was an efficient approach for sample preparation and isotopic labeling of the chimeric R3/I5 peptide. This approach could also be extended to the preparation of other relaxin family peptides in future studies.
"Coagulants used for cheese manufacturing come from different sources, such as microorganisms, plants, animals, and recombinant protein expression, and each source has different characteristics determining their applications (Jiang et al. 2012). The methylotrophic yeast Komagataella (Pichia ) pastoris is extensively used for the expression of heterologous proteins (Sreekrishna and Kropp 1996) because it exhibits several advantages over other microbial systems like Escherichia coli: rapid growth and high-density fermentations (Cregg et al. 2009) and the ability to perform many of the eukaryotic posttranslational protein modifications like folding, proteolytic processing , glycosylation, and disulfide bond formation (Cereghino et al. 2002). "
[Show abstract][Hide abstract] ABSTRACT: The use of agroindustrial wastes not only decreases bioprocesses and disposal costs but also contributes to the upgrading of the residues. An active recombinant methanol-inducible bovine chymosin has been expressed in our laboratory in the yeast Komagataella pastoris, and grape pomace extracts (GRE) were proposed as a convenient C-energy source for the biomass production of the genetically engineered strain. Carbon and nitrogen sources, growth factors, and initial pH conditions were selected by classical methodology; thereafter, growth conditions optimization was performed using statistical designed experiments (DoEs). In the presence of (in g·L−1) 67.0 monosaccharides (glucose and fructose) from GRE, 5.0 (NH4)2SO4, and 10.0 sugar cane molasses (CMz), a yield of 20.0 g·L−1 cell dry weight (CDW) was obtained aerobically after 60 h incubation at 28°C and pH 4.0. Applying a fed-batch strategy with methanol:sorbitol as the enzyme inducers, a chymosin production of 8.53 International Milk Clotting Units (IMCU) per mg protein was obtained in the supernatant. The results presented show that through a statistical design, a simple, cheap, and easy to prepare culture medium could be developed using two agroindustrial derivatives (GRE and CMz) to obtain a higher value added product.