Expression of Recombinant Genes in the Yeast Pichia pastoris

James M. Cregg1, Ilya Tolstorukov1, Anasua Kusari2, Anthony Jay Sunga2, Knut Madden1, Thomas Chappell1

1 Biogrammatics, Inc., Carlsbad, California, 2 Keck Graduate Institute of Applied Life Sciences, Claremont, California
Publication Name:  Current Protocols Essential Laboratory Techniques
Unit Number:  Unit 13.2
DOI:  10.1002/9780470089941.et1302s04
Online Posting Date:  December, 2010
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Abstract

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. © 2010 by John Wiley & Sons, Inc.

Keywords: recombinant proteins; foreign gene expression; heterologous genes

     
 
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Table of Contents

  • Overview and Principles
  • Protocols
  • Basic Protocol 1: Transformation by Electroporation
  • Alternate Protocol 1: Rapid Preparation of Electrocompetent Cells
  • Basic Protocol 2: Selection of Strains with Multiple Copies of an Expression Vector: Post‐Translational Vector Amplification (PTVA)
  • Basic Protocol 3: Small‐Scale Expression of Recombinant Genes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Transformation by Electroporation

  Materials
  • E. coli containing expression plasmid with foreign DNA of interest
  • Restriction enzyme for linearizing vector
  • P. pastoris strain to be transformed (Invitrogen)
  • Yeast‐extract Peptone Dextrose (YPD) liquid medium (see recipe)
  • YPD liquid medium containing 200 mM HEPES, pH 8.0
  • 1 M dithiothreitol (DTT)
  • 1 M sorbitol
  • Appropriate selective agar plates: for zeocin or other antibiotic selection, selective medium is composed of YPD plus 100 µg/ml zeocin or other antibiotic; for auxotrophic selections, use 0.67% yeast nitrogen base (YNB with ammonium sulfate but without amino acids and dextrose) plus 2% glucose
  • 30°C shaking incubator
  • 2.8‐liter Fernbach culture flasks
  • Refrigerated centrifuge
  • Sterile 250‐ml centrifuge tubes
  • End‐over‐end rotator
  • Sterile 2‐mm electroporation cuvettes
  • Electroporation instrument (e.g., BTX Electro Cell Manipulator 600, http://btxonline.com; Bio‐Rad Gene Pulser; or Electroporator II, Invitrogen,)
  • Additional reagents and equipment for protein expression in P. pastoris (Higgins et al., ) and determination of optical density of bacterial culture (unit 4.2)
NOTE: All solutions should be autoclaved except for the DTT and HEPES solutions, which should be filter sterilized.

Alternate Protocol 1: Rapid Preparation of Electrocompetent Cells

  • BEDS solution (see recipe) with and without 0.1 M DTT
  • 250‐ to 500‐ml Erlenmeyer flasks

Basic Protocol 2: Selection of Strains with Multiple Copies of an Expression Vector: Post‐Translational Vector Amplification (PTVA)

  Materials
  • Vector containing a zeocin‐resistance selection marker (e.g., pPICZ series vectors; Invitrogen)
  • YPD agar (see recipe) with 100 µg/ml, 500 µg/ml, or 1 to 2 mg/ml zeocin
  • “Six‐cutter” restriction enzyme (e.g., BglI)
  • Additional reagents and equipment for transformation and selection of P. pastoris ( protocol 1), agarose gel electrophoresis (unit 7.2), and Southern blotting and hybridization (unit 8.2)

Basic Protocol 3: Small‐Scale Expression of Recombinant Genes

  Materials
  • Human serum albumin (HSA)–secreting strain of P. pastoris (available from the authors; ) and negative (wild‐type strain) control
  • Buffered methanol minimal complex (BMMY) medium (see recipe)
  • Methanol
  • 6× SDS sample buffer (unit 7.3)
  • β‐lactamase‐producing P. pastoris strains (available from the authors; ; see protocol introduction above)
  • Yeast‐extract Peptone Dextrose (YPD) liquid medium (see recipe)
  • YNB glucose medium (see recipe)
  • YNB methanol medium (see recipe)
  • Glass beads (0.45‐mm)
  • Breaking buffer: 25 mM Tris⋅Cl, pH 7.5 (unit 3.3), ice cold
  • Nitrocefin substrate solution (see recipe)
  • Nitrocefin (EMD Biosciences)
  • 500‐ml Erlenmeyer flasks, sterile
  • 30°C shaking incubator
  • Spectrophotometer
  • Centrifuge
  • 50‐ml sterile shake flasks (Kimax Erlenmeyer flasks, cat no. S43013)
  • Multi‐sample vortex head (Disruptor Genie, Scientific Industries, Inc., http://www.scientificindustries.com/; optional)
  • Additional reagents and equipment for protein expression in P. pastoris (Higgins et al., ), determination of optical density of bacterial culture (unit 4.2), SDS‐PAGE (unit 7.3), staining of gels (unit 7.4), and determining protein concentration (unit 2.2)
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Figures

  •   FigureFigure 13.2.1 Photograph of an agar plate containing transformed P. pastoris colonies resulting from a successful electroporation experiment. Approximately 1 × 106 cells were spread on this plate.
  •   FigureFigure 13.2.2 Selected P. pastoris strains after undergoing the PTVA enrichment process. Key is shown at the right. Strains shown are: untransformed (UT) wild type (WT) strain (NRRL‐Y11430, not resistant to zeocin); strains 6,7,11,13,14,15 are highly enriched strains and therefore highly resistant to high concentrations of zeocin.
  •   FigureFigure 13.2.3 Southern blot showing genomic DNA from selected P. pastoris strains transformed with vector pPICZB‐β‐lactamase. Labeled DNA fragments containing sequences from the 5′ AOX1 promoter (A) and labeled DNA fragments containing sequences from the zeocin gene (B) were used as probes to compare and validate whole‐plasmid amplification. Lanes contain genomic DNA digested with BglI from each strain. Strains shown are: untransformed control (UT); “enriched” strains are denoted by the parent number followed by “–1”; while “pre‐enriched” equivalent strains are denoted by a number only.
  •   FigureFigure 13.2.4 Flow diagram of small‐scale P. pastoris expression.

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Literature Cited

Literature Cited
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   Clare, J.J., Romanos, M.A., Rayment, F.B., Rowedder, J.E., Smith, M.A., Payne, M.M., Sreekrishna, K., and Henwood, C.A. 1991. Production of mouse epidermal growth factor in yeast: High‐level secretion using Pichia pastoris strains containing multiple gene copies. Gene 105:205‐212.
   Cregg, J.M. (ed.) 2007. Methods in Molecular Biology: Pichia Protocols, 2nd Edition, Humana Press, TotowaNew, Jersey.
   Cregg, J.M. and Madden, K.R. 1988. Development of the methylotrophic yeast, Pichia pastoris, as a host system for the production of foreign proteins. Dev. Ind. Microbiol. 29:33‐41.
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   Tschopp, J.F., Brust, P.F., Cregg, J.M., Stillman, C.A., and Gingeras, T.R. 1987. Expression of the lacZ gene from two methanol‐regulated promoters in Pichia pastoris. Nucleic Acids Res. 15:3859‐3875.
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