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Overview of Protein Expression in Pichia pastoris

David R. Higgins¹

¹Invitrogen Corporation, San Diego, California

Publication Name:

Current Protocols in Protein Science

Unit Number:

Unit 5.7

DOI:

10.1002/0471140864.ps0507s02

Online Posting Date:

May, 2001

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Abstract

Pichia pastoris is a methylotrophic yeast and can be used as a heterologous expression system. This microorganism is as easy to manipulate as Escherichia coli, but has many of the advantages of eukaryotic expression (e.g., protein processing, folding, and post-translational modifications), and it is faster, easier, and cheaper to use than other eukaryotic expression systems, such as baculovirus or mammalian tissue culture. It also generally yields higher expression levels. This overview discusses important considerations for the use of Pichia pastoris, including strains for expression, expression plasmids, transformation by integration, and post-translational modifications. Examples of expression are given and finally, legal issues regarding patent rights for heterologous protein expression in Pichia pastoris are described.

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Unit Introduction
General Characteristics of Pichia pastoris
Heterologous Protein Expression
Commentary
Literature Cited
Figures
Tables

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Figures

Figure 5.7.1

Generic plasmid for heterologous protein expression in Pichia pastoris. Elements present in all expression plasmids are shown in dark boxes, optional elements present in some plasmids are shown in gray boxes. Abbreviation: TT, transcriptional termination sequence.

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Figure 5.7.2

Gene replacement. A gene replacement event at the AOX1 locus by a plasmid fragment carrying an “expression cassette” consisting of an AOX1 promoter–driven gene of interest and the HIS4 selectable marker allows for selection of His⁺ prototrophs using a his4 Pichia strain. The event represents a double cross-over between two linear DNA molecules. Double-stranded ends of DNA molecules act to target and stimulate homologous recombination. The result is a loss of the AOX1 locus (the strain becomes Mut^S) and the gain of P_AOX1-gene of interest-HIS4 (gene of interest⁺ His⁺). Abbreviations: ORF, open reading frame; TT, transcriptional termination sequence.

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Figure 5.7.3

Gene insertion. A gene insertion event 3¢ of AOX1 in the genome by the plasmid carrying an expression cassette allows for selection of His⁺ prototrophs using a his4 Pichia strain. The event represents a single cross-over event between circular and linear DNA molecules. Recombination takes place within regions of homology between the plasmid and the genome. The result is an insertion of the plasmid 3¢ to the intact AOX1 locus (the strain remains Mut⁺) and the gain of P_AOX1-gene of interest-HIS4 (gene of interest⁺ His⁺). This event also could have taken place between the 5¢ AOX1 regions on the plasmid and genome with the resulting insertion positioned 5¢ to an intact AOX1 locus. Abbreviations: ORF, open reading frame; TT, transcriptional termination sequence.

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Figure 5.7.4

Gene insertion in his4. A gene insertion event within the his4 locus of the genome by the plasmid carrying an expression cassette allows for selection of His⁺ prototrophs using a his4 Pichia strain. The event consists of a single cross-over between circular and linear DNA molecules, in which recombination takes place between the HIS4 locus on the plasmid and the his4 locus in the genome. The result is an insertion of the plasmid between duplicated copies of the HIS4/his4 genes, one still mutant and the other wild-type (the strain remains Mut⁺, and becomes gene of interest⁺ His⁺). Abbreviations: TT, transcriptional termination sequence.

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Figure 5.7.5

Multiple insertion events. Multiple insertions of an expression plasmid 3¢ to an otherwise intact AOX1 locus can result from multimer formation prior to a single insertion event or from multiple sequential insertion events at the same locus. These events can also take place between the 5¢ AOX1 regions on the plasmid and genome, with the resulting insertions positioned 5¢ to an intact AOX1 locus. Multiple events (two or more plasmids integrated) occur spontaneously, although infrequently, accounting for 1% to 10% of all His⁺ transformants. Abbreviations: ORF, open reading frame; TT, transcriptional termination sequence.

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Figure 5.7.6

Multiple insertions with a Kan^r-containing plasmid. Multiple insertions of an expression plasmid 3¢ to an otherwise intact AOX1 locus can result from multimer formation prior to a single insertion event or from multiple sequential insertion events at the same locus. These events can also occur between the 5¢ AOX1 regions on the plasmid and genome, with the resulting insertions 5¢ to an intact AOX1 locus. Each expression cassette introduces a copy of the bacterial Kan^r gene, which confers resistance to G418; multiple copies make recombinant strains hyperresistant to G418, which phenotypically identifies strains carrying multiple copies of the expression cassette. Abbreviations: ORF, open reading frame; TT, transcriptional termination sequence.

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Figure 5.7.7

In vitro multimer formation. By this scheme, multiple copies of P_AOX1-gene of interest are ligated together in a plasmid in vitro, and linked to a single HIS4 gene in an expression cassette. Transformation of Pichia with these in vitro–formed multimers is used as a tool to increase the frequency with which multiple-copy expression cassette recombinant strains are generated. Abbreviations: OEF, open reading frame; TT, transcriptional termination sequence.

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Figure 5.7.8

Flow chart for the generation of recombinant strains of Pichia pastoris.

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Literature Cited

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Key References
	Cregg et al., 1993. See above.
Good overview of Pichia pastoris as an expression system with a discussion of glycosylation of expressed proteins.
	Scorer et al., 1993. See above.
Describes in detail the advantages of intracellular versus secreted expression, the effect of multiple-copy expression cassettes on expression levels, and an overall approach to optimizing expression levels.
	Scorer et al., 1994. See above.
Details the generation of multiple-copy expression cassette recombinant strains using the bacterial Kan^r gene and screening for hyperresistance to G418.