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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Biotechniques. Author manuscript; available in PMC Aug 8, 2008.
Published in final edited form as:
PMCID: PMC2504082
NIHMSID: NIHMS58298

Condensed protocol for competent cell preparation and transformation of the methylotrophic yeast Pichia pastoris

The methylotrophic yeast Pichia pastoris has gained widespread acceptance as a system of choice for heterologous protein expression in part because of the simplicity of techniques required for its molecular genetic manipulation (1). Several different procedures are available for introducing DNA into P. pastoris—spheroplast generation (2), electroporation (3), alkali cation (3,4), or polyethylene glycol (PEG) treatment (5). Here we describe a condensed protocol for cell preparation and transformation that works reliably with either auxotrophic markers or antibiotic selection.

The introduction of exogenous DNA into an organism requires two steps: (i) the preparation of competent cells for DNA uptake and (ii) the transformation of the cells with the DNA. Transformation of P. pastoris by electroporation is a quick procedure. However, preparation of conventional electroporation-competent cells requires hours of work involving several washes, incubations, and centrifugations. In contrast, competent cell preparation for the heat-shock method is short, but transformation requires approximately 2 h (4). The heat-shock procedure gives approximately 100-fold lower transformation efficiency than electroporation with plasmids containing auxotrophic marker genes such as HIS4. Additionally, the selection of zeocin-resistant transformants using the heat-shock transformation protocol does not work reliably.

We have modified the preparation of competent cells from the heat-shock procedure (5) and combined it with transformation by electroporation (3) to yield a condensed protocol that works consistently with auxotrophic markers or antibiotic selection. The main modification of the heat-shock procedure is the addition of a step in which P. pastoris cells are incubated in an optimized concentration of dithiothreitol (DTT). The cells prepared by this “hybrid” method are then electroporated using the same parameters as conventional electroporation.

Transformation efficiencies using the condensed protocol are comparable to the conventional electroporation procedure using auxotrophic markers but are approximately 20-fold lower using the zeocin resistance marker. However, the condensed protocol provides sufficient transformants, including multicopy integrants, for protein expression studies and has several advantages over the conventional electroporation and heat-shock methods. Table 1 compares the steps in cell preparation and transformation for conventional electroporation, heat shock, and our condensed protocol. Compared to the heat-shock method, the condensed protocol requires less time for the transformation step and provides much higher transformation efficiencies. Compared to the electroporation procedure, the new procedure saves both reagents and time during cell preparation. In addition, the fewer number of steps during the cell preparation of the condensed protocol reduce the chance of contamination of competent yeast cells. Furthermore, unlike the electroporation cell preparation procedure, our condensed protocol does not require a large, refrigerated centrifuge. We use a small, nonrefrigerated centrifuge capable of spinning six 50-mL conical tubes at a time, enabling us to quickly prepare competent cells of six different strains simultaneously. This is significant because some P. pastoris strains (i.e., protease deficient, methanol utilization deficient) can express a given protein more efficiently than others, and it is often necessary to transform the same expression plasmid into various strains to determine empirically which strain gives the highest expression. Thus, the condensed protocol (Table 2) enables a researcher to prepare and transform multiple samples of highly competent P. pastoris cells in a short time with minimal equipment or effort.

Table 1
Comparison of Transformation Protocols
Table 2
Condensed Protocol: Preparation of Competent Cells and Transformation

Using HIS4-based and zeocin-based plasmids based on typical P. pastoris cloning vectors, pHILA1 and pPICZB (Invitrogen, Carlsbad, CA, USA), and strains such as JC100 (wild-type) or GS115 (his4), we obtained the results summarized in Table 3. These results are average transformation efficiencies calculated from at least five separate transformations.

Table 3
Transformation Efficiencies Method

The condensed protocol utilizes the most efficient portions of the electroporation and heat-shock transformation protocols to yield a procedure for P. pastoris cells that produces high transformation efficiencies while saving time, effort, and reagents.

ACKNOWLEDGMENTS

The authors would like to thank all members of the Lin-Cereghino laboratory for their support. This work was funded by undergraduate research funds from the University of the Pacific and National Institutes of Health (NIH)-AREA grant no. GM65882 to J.L.-C. and G.P.L.-C.

Footnotes

COMPETING INTERESTS STATEMENT

The authors declare no competing interests.

REFERENCES

1. Lin Cereghino J, Cregg JM. Heterologous protein expression in the methylotrophic yeast Pichia pastoris. FEMS Microbiol. Rev. 2000;24:45–66. [PubMed]
2. Cregg JM, Barringer KJ, Hessler AY, Madden KR. Pichia pastoris as a host system for transformations. Mol. Cell. Biol. 1985;5:3376–3385. [PMC free article] [PubMed]
3. Cregg JM, Russell KA. Transformation methods. In: Higgins DR, Cregg JM, editors. Methods in Molecular Methods Molecular Biology. Vol. 103. Humana Press; Totowa, NJ: 1998. pp. 27–39. Pichia Protocols. [PubMed]
4. Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J. Bacteriol. 1983;153:163–168. [PMC free article] [PubMed]
5. Dohmen RJ, Strasser AWM, Höner CB, Hollenberg CP. An efficient transformation procedure enabling long-term storage of competent cells of various yeast genera. Yeast. 1991;7:691–692. [PubMed]
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