Culture of Yeast for the Production of Heterologous Proteins

Michael A. Romanos1, Jeffrey J. Clare1, Crawford Brown2

1 Wellcome Research Laboratories, Beckenham, 2 British Bio‐Technology Ltd., Cowley
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 5.8
DOI:  10.1002/0471140864.ps0508s02
Online Posting Date:  May, 2001
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Abstract

This unit describes culture of the yeast strains Saccharomyces cerevisiae and Pichia pastoris for the production of foreign proteins. The protocols listed here for S. cerevisiae are for three widely used types of promoter: galactose‐regulated (GAL1, GAL7, GAL10), glucose‐repressible (e.g., ADH2), and constitutive glycolytic (e.g., PGK or GAPDH). Minor variations to each can be made depending on the selection system used. The P. pastoris expression system uses integrating vectors with the methanol‐regulated AOX1 promoter and HIS4 selection marker; although transformants are stable, they are generally grown in minimal selective medium. Methods are described for small‐scale S. cerevisiae and P. pastoris cultures and also for high‐density fermentations with these yeasts. A simple feeding strategy based on calculated feed rates is provided for S. cerevisiae and yields cell densities of 10 to 30 g/liter. In contrast, with P. pastoris, basic fermenter equipment is used to obtain extremely high‐density cultures (e.g., 130 g/liter). Finally, a describes small‐scale preparation of protein extracts.

     
 
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Table of Contents

  • Basic Protocol 1: Small‐Scale Expression Using S. cerevisiae Galactose‐Regulated Vectors
  • Alternate Protocol 1: Expression Using Glucose‐Repressible ADH2 Vectors
  • Alternate Protocol 2: Expression Using Vectors with Glycolytic Gene Promoters
  • Basic Protocol 2: Small‐Scale Expression in Pichia pastoris
  • Basic Protocol 3: Large‐Scale Expression Using S. cerevisiae Galactose‐Regulated Vectors
  • Basic Protocol 4: Large‐Scale Expression Using Pichia pastoris
  • Support Protocol 1: Small‐Scale Preparation of Protein Extracts
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Small‐Scale Expression Using S. cerevisiae Galactose‐Regulated Vectors

  Materials
  • YP broth (see recipe) containing 2% (w/v) raffinose or YNB medium (see recipe) for auxotrophic selection
  • 50 mg/ml G418 (100×) or supplements for auxotrophic selection, as required
  • Saccharomyces cerevisiae transformed with a galactose‐regulated expression vector and plated on selective medium (see unit 5.6 and Ausubel et al., , Chapter 13)
  • 20% (w/v) galactose (filter sterilize and store at room temperature)
  • H 2O, sterile
  • Plastic 25‐ml universal containers, sterile (e.g., Nunc)
  • Heraeus omnifuge centrifuge and 2250 rotor (or equivalent)
  • 15‐ml polypropylene snap‐top tubes
  • Centricon concentrator (Amicon; also see unit 4.4)

Alternate Protocol 1: Expression Using Glucose‐Repressible ADH2 Vectors

  • YP broth (see recipe) containing 8% (w/v) glucose
  • Saccharomyces cerevisiae transformed with a glucose‐repressible ADH2 expression vector and plated on selective medium (see unit 5.6 and Ausubel et al., , Chapter 13)
  • YP broth (see recipe) containing either 2% (w/v) raffinose and 2% (v/v) ethanol or 1% (w/v) glucose
  • 100‐ml Erlenmeyer flask, sterile
  • Diastix glucose test strips (Bayer Diagnostics; optional)

Alternate Protocol 2: Expression Using Vectors with Glycolytic Gene Promoters

  • YNB medium (see recipe) containing 2% (w/v) glucose and the required supplements
  • Saccharomyces cerevisiae transformed with an expression vector containing a glycolytic gene promotor and plated on selective medium (see unit 5.6 and Ausubel et al., , Chapter 13)

Basic Protocol 2: Small‐Scale Expression in Pichia pastoris

  Materials
  • YNB medium (see recipe) containing 2% (w/v) glycerol
  • Pichia pastoris transformed with an expression vector and plated on selective medium (see unit 5.7 and Ausubel et al., , Chapter 13)
  • 0.002 mg/ml biotin (500×; filter sterilize and store <1 year at 4°C)
  • H 2O, sterile
  • YNB medium (see recipe) containing 1% (v/v) methanol
  • Plastic 25‐ml universal containers, sterile
  • Heraeus omnifuge centrifuge and 2250 rotor (or equivalent)
  • 100‐ml Erlenmeyer flasks, sterile
  • 15‐ml polypropylene snap‐top tubes

Basic Protocol 3: Large‐Scale Expression Using S. cerevisiae Galactose‐Regulated Vectors

  Materials
    YNB medium (see recipe) containing 1% (w/v) glucose and appropriate supplements
  • Saccharomyces cerevisiae transformed with a galactose‐regulated expression vector and plated on selective medium (see unit 5.6 and Ausubel et al., , Chapter 13)
  • BSSM fermenter medium (see recipe)
  • 20% (w/v) glucose
  • 20% (w/v) galactose
  • 3 M phosphoric acid
  • 3 M sodium hydroxide
  • Antifoam (Dow Corning M30 antifoam)
  • 250‐ml and 2‐liter Erlenmeyer flasks, sterile
  • Benchtop fermenter, 5‐liter total volume, with data logging and MENTOR control system (Life Science Laboratories)
  • Electronic balance (Mettler) with RS232 communications
  • Sorvall RC‐3B centrifuge and H6000A rotor (or equivalent)

Basic Protocol 4: Large‐Scale Expression Using Pichia pastoris

  Materials
  • YNB medium (see recipe) containing 2% (v/v) glycerol
  • Pichia pastoris transformed with an expression vector and plated on selective medium (see unit 5.7 and Ausubel et al., , Chapter 13)
  • 5× basal salts solution (see recipe)
  • PTM 1 solution (see recipe)
  • Glycerol/PTM 1: 12 ml PTM 1 solution/liter 50% (v/v) glycerol
  • 50% (v/v) ammonium hydroxide
  • Methanol/PTM 1: 12 ml PTM 1 solution/liter 100% methanol
  • Fermenter equipped with monitors or controls for pH, temperature, dissolved O 2, airflow, and stirring speed
  • Erlenmeyer flask, sterile

Support Protocol 1: Small‐Scale Preparation of Protein Extracts

  Materials
  • Cell pellet from an induced culture of transformed yeast cells
  • Protein extraction buffer (see recipe), ice‐cold
  • 5× protease inhibitor cocktail (see recipe)
  • 15‐ml polypropylene snap‐top tubes
  • 0.45‐mm glass beads, acid washed (see recipe)
  • Platform vortex mixer (e.g., IKA‐Vibrax‐VXR with test tube rack)
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Figures

Videos

Literature Cited

   Aiba, S., Nagai, S., and Nishizawa, Y. 1976. Fed‐batch culture of Saccharomyces cerevisiae: A perspective of computer control to enhance the productivity of Baker's yeast cultivation. Biotechnol. Bioeng. 18:1001‐1016.
   Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A., and Struhl, K. (eds.) 1995. Current Protocols in Molecular Biology. John Wiley & Sons, New York.
   Bock Gu, M., Jung, K.‐H, Park, M.N., Shin, K.S., and Kim, K.H. 1989. Production of HbsAg by growth‐rate control with recombinant Saccharomyces cerevisiae in fed‐batch. Biotech. Lett. 11:1‐4.
   Clare, J.J., Rayment, F.B., Ballantine, S.P., Sreekrishna, K., and Romanos, M.A. 1991a. High‐level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Bio/Technology 9:455‐460.
   Clare, J.J., Romanos, M.A., Rayment, F.B., Rowedder, J.E., Smith, M.A., Payne, M.M., Sreekrishna, K., and Henwood, C.A. 1991b. 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., Tschopp, J.F., Stillman, C., Siegel, R., Akong, M., Craig, W.S., Buckholz, R.G., Madden, K.R., Kellaris, P.A., Davies, G.R., Smiley, B.L., Cruze, J., Torregrossa, R., Velicelebi, G., and Thill, G.P. 1987. High‐level expression and efficient assembly of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris. Bio/Technology 5:479‐485.
   Cregg, J.M., Vedvick, T.S., and Raschke, W.C. 1993. Recent advances in the expression of foreign genes in Pichia pastoris. Bio/Technology 11:905‐910.
   Fieschko, J.C., Egan, K.M., Ritch, T., Koski, R.A., Jones, M., and Bitter, G.A. 1987. Controlled expression and purification of human immune interferon from high‐cell‐density fermentations of Saccharomyces cerevisiae. Biotechnol. Bioeng. 29:113‐1121.
   Hsieh, J.‐H., Shih, K.‐Y., Kung, H.‐F., Shiang, M., Lee, L.‐Y., Meng, M.‐H., Chang, C.‐C., Lin, H.‐M. Shih, S.‐C., Lee, S.‐Y., Chow, T.‐Y., Feng, T.‐Y., Kuo, T.‐T., and Choo, K.‐B. 1988. Controlled fed‐batch fermentation of recombinant Saccharomyces cerevisiae to produce hepatitis B surface antigen. Biotechnol. Bioeng. 32:334‐340.
   Laroche, Y., Storme, V., De Meutter, J., Messens, J., and Lauwereys, M. 1994. High‐level secretion and very efficient isotopic labeling of tick anticoagulant peptide (TAP) expressed in the methylotrophic yeast, Pichia pastoris. Bio/Technology 12:1119‐1124.
   Pirt, S.J. 1985. Batch cultures with substrate feeds. In Principles of Microbe and Cell Cultivation, pp. 211‐222. Blackwell Scientific, Oxford.
   Romanos, M.A., Clare, J.J., Beesley, K.M., Rayment, F.B., Ballantine, S.P., Makoff, A.J., Dougan, G., Fairweather, N.F., and Charles, I.G. 1991. Recombinant Bordetella pertussis pertactin (P69) from the yeast Pichia pastoris: High‐level production and immunological properties. Vaccine 9:901‐906.
   Romanos, M.A., Scorer, C.A., and Clare, J.J. 1992. Foreign gene expression in yeast: A review. Yeast 8:423‐488.
   Scorer, C.A., Clare, J.J., McCombie, W.R., Romanos, M.A., and Sreekrishna, K. 1994. Rapid selection using G418 of high‐copy‐number transformants of Pichia pastoris for high‐level foreign gene expression. Bio/Technology 12:181‐184.
   Sonnlietner, B. and Kappeli, O. 1986. Growth of Saccharomyces cerevisiae is controlled by its limited respiratory capacity: Formation and verification of a hypothesis. Biotechnol. Bioeng. 28:927‐937.
   Sreekrishna, K., Nelles, L., Potenz, R., Cruze, J., Mazzaferro, P., Fish, W., Motohiro, F., Holden, K., Phelps, D., Wood, P., and Parker, K. 1989. High‐level expression, purification, and characterization of recombinant human tumour necrosis factor synthesized in the methylotrophic yeast Pichia pastoris. Biochemistry 28:4117‐4125.
   Thill, G.P., Davis, G.R., Stillman, C., Holtz, G., Brierley, R., Engel, M., Buckholtz, R., Kinney, J., Provow, S., Vedvick, T., and Seigel, R.S. 1990. Positive and negative effects of multi‐copy integrated expression vectors on protein expression in Pichia pastoris. In Proceedings of the 6th International Symposium on Genetics of Microorganisms, Vol. II (H. Heslot, J. Davies, J. Florent, L. Bobichon, G. Durand, and L. Penasse, eds.) pp. 477‐498. Société Francaise de Microbiologie, Paris.
   Wang, H.Y., Cooney, C.L., and Wang, D.I. 1979. Computer control of bakers' yeast production. Botechnol. Bioeng. 21:975‐995.
Key References
   Cregg et al., 1993. See above.
  An excellent review of P. pastoris expression technology.
  McNeil, B. and Harvey, L.M. eds. 1990. Fermentation: A practical approach. Oxford University Press, Oxford.
  A detailed laboratory manual covering all aspects fermentation, especially useful to those new to fermentation technology.
   Romanos et al., 1992. See above.
  A detailed review covering all aspects of yeast expression, including vector systems, factors affecting foreign gene expression, secretion, alternative yeasts (including P. pastoris), and fermenter scale‐up.
   Romanos, M.A., Scorer, C.A., and Clare, J.J. 1995. Expression of cloned genes in yeast. In DNA Cloning 2, Expression Systems: A Practical Approach (D.M. Glover and B.D. Hames, eds.) pp. 123‐167. IRL Press, Oxford.
  A detailed practical review of the basic strategies and techniques for high‐level expression of genes in yeast including approaches for diagnosing and solving problems.
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