High-Level Expression of Tetanus Toxin Fragment C in Pichia Pastoris Strains Containing Multiple Tandem Integrations of the Gene

Abstract

We have used the methylotrophic yeast, Pichia pastoris, to express high levels of tetanus toxin fragment C, a potential subunit vaccine against tetanus. In high biomass fermentations fragment C was induced to 27% of total cell protein or about 12 g/l of culture. The purified protein was as effective as native fragment C in immunizing mice. In order to optimize fragment C production, we have examined the parameters affecting foreign gene expression in Pichia. The level of expression was found to be largely independent of the site of chromosomal integration of the gene (AOX1 or HIS4), the type of integrant (insertion or transplacement), and the methanol utilisation phenotype of the host strain (Mut+ or Muts). The most important factor in obtaining high levels was the presence of multiple integrated copies of the fragment C expression cassette. Multicopy clones could be isolated from transformations using DNA fragments targeted for single-copy transplacement into the chromosome. These multicopy transformants were surprisingly stable over multiple generations during growth and induction in high cell density fermentations. Analysis of chromosomal DNA from these clones suggests that they arose by circularization of the transforming DNA fragment in vivo followed by multiple insertion into the chromosome via repeated single crossover recombination, in addition to the expected transplacement event. We have found this to be a general phenomenon and have used these multicopy "transplacement" clones to obtain high-level expression of several other foreign genes in Pichia.

Heterologous protein production using the Pichia pastoris expression system

“…This range does not affect the growth significantly, which allows considerable freedom in adjusting the pH to one that is not optimal for a problem protease. Different pH values were found to be optimal from the point of view of a recombinant protein's stability: pH 6.0 was optimal in production of recombinant mouse epidermal factor and human serum albumin [25,59] and pH 3.0 was optimal in production of insulin-like growth factor-I and cytokine growth-blocking peptide (50 mg/l) [13,60].…”

Development of vaccines for prevention of botulism

“…Clare and coworkers [59] reported yields of 12 g/L of TeNT(H C ) when expressed in P. pastoris. Using P. pastoris would also eliminate the potential problems associated with inclusion bodies formed during expression in E. coli, as well as the need to remove endotoxin.…”

“…Because of rare codons [60], and high adenine and thymine (A+T)-rich base compositions [61] found in the naturally-occuring clostridial DNA, it was necessary to construct synthetic genes [57,59,61] to eliminate these impediments to optimal expression in heterologous expression systems such as E. coli and yeast. We prepared synthetic genes for the H C of BoNT with codon usage specified by highly expressed genes in E. coli and yeast.…”

“…The expression cassette was integrated into the chromosomal alcohol oxidase of P. pastoris strain GS115 [59]. Yeast transformants expressing the selectable markers, histidine dehydrogenase [70] and aminoglycoside phosphotransferase 3' I [68], were isolated.…”

Direct selection of Pichia pastoris expression strains using new G418 resistance vectors

“…Another way of generating multicopy strains is simply to screen large numbers of colonies for spontaneously occurring transformants with higher gene dosage or higher product expression (Clare et al, 1991a;Romanos et al, 1991;Wung and Gascoigne, 1996). However, at this time only two dominant markers, the bacterial Sh ble (Zeocin r ) or BSD (blasticidin r ) genes, can be used to directly select transformants and screen for high-copy strains on increasingly higher concentrations of Zeocin or blasticidin (Miles et al, 1998;Romanos et al, 1991).…”