Expression Vector and Identification of an Expression Strain

Polymerase chain reaction (PCR) techniques were used to identify two alcohol oxidase-encoding genes in P. methanolica: AUG1 and AUG2 (alcohol utilization gene; Raymond et al, 1998). The alcohol oxidases encoded by these genes share 83% identity with one another. Auglp shares 84% identity with Moxlp from Hansenula poly-morpha, 72% identity with Aodlp from Candida boidini, and 69% identity with Aoxlp from P. pastoris. Three lines of evidence indicate that Auglp is the dominant alcohol oxidase in P. methanolica and therefore the AUG 1 promoter is appropriate choice to drive recombinant protein expression. First, amino-terminal sequence analysis of the alcohol oxidase induced by methanol in a high-cell density fermentation demonstrated that only Auglp was present. Second, an auglA mutant grows poorly in minimal methanol broth whereas an aug2A mutant has a wild-type growth phenotype (an auglh aug2A double mutant cannot grow at all). Finally, the levels of recombinant protein expression driven by the AUG1 promoter in P. methanolica are comparable to the expression levels achieved with the AOX1 promoter in P. pastoris (see later).

A protein expression vector, pCZR134, that utilizes the AUG1

transcriptional control elements and the ADE2 selectable marker was assembled into a pUC19 vector (Fig. 2). Protein-coding sequences can be cloned into unique £coRI, BamHl, or Spel sites. The EcoRI site is positioned at nucleotide +1 of the alcohol oxi-dase-coding region. In limited studies, placement of the cloning sites 5' of the alcohol oxidase-coding region at position -50 or -100 relative to the alcohol oxidase start codon had no noticeable influence on protein expression levels (C. K. Raymond, unpublished). For transformation into P. methanolica, the expression cassette is liberated from the pUC backbone as a linear DNA fragment by digestion with either Notl or Sfil.

To identify an expression strain, stable Ade+ transformants are routinely gridded to minimal methanol plates, overlayed with nitrocellulose, and grown for 2-3 days. To detect intracellular proteins, colonies that adhere to the filter can be lysed using a simple protocol (Wuestehube et al., 1996). Following lysis, or in the case of secreted proteins, the filters are then probed with an antibody that recognizes the recombinant protein using standard Western blot techniques. To date, most transformants have been AUG1, consistent with a mechanism whereby expression cassettes integrate into the host genome via circularization followed by integration. Disruption of the AUG1 gene by direct, double crossover

Figure 2 The P. methanolica protein expression vector, pCZR134, was made as described in Raymond et al. (1998). cDNAs to be expressed can be cloned into unique EcoRI, BamHl, and/or Spel sites. The scale shown is in kilobases, and the empty expression vector is 8.1 kb. Reproduced with permission from Raymond, C. K., Bukowski, T., Holderman, S. D., Ching, A. F. T., Vanaja, E., and Stamm, M. R. (1998). Development of the methylotrophic yeast, Pichia methanolica, for the expression of the 65-kilodalton isoform of human glutamate decarboxylase. Yeast 14, 11-23. Copyright © John Wiley & Sons Limited.

Figure 2 The P. methanolica protein expression vector, pCZR134, was made as described in Raymond et al. (1998). cDNAs to be expressed can be cloned into unique EcoRI, BamHl, and/or Spel sites. The scale shown is in kilobases, and the empty expression vector is 8.1 kb. Reproduced with permission from Raymond, C. K., Bukowski, T., Holderman, S. D., Ching, A. F. T., Vanaja, E., and Stamm, M. R. (1998). Development of the methylotrophic yeast, Pichia methanolica, for the expression of the 65-kilodalton isoform of human glutamate decarboxylase. Yeast 14, 11-23. Copyright © John Wiley & Sons Limited.

transplacement with the expression cassette appears to be a relatively rare event. Most (-90%) transformants appear to make some level of recombinant protein. Strategies to evaluate protein expression and to identify high-yield expression strains are considered next.

Cure Your Yeast Infection For Good

Cure Your Yeast Infection For Good

The term vaginitis is one that is applied to any inflammation or infection of the vagina, and there are many different conditions that are categorized together under this ‘broad’ heading, including bacterial vaginosis, trichomoniasis and non-infectious vaginitis.

Get My Free Ebook


Post a comment