HETEROLOGOUS PROTEIN EXPRESSION IN THE METHYLOTROPHIC YEAST PICHIA PASTORIS PDF

Corresponding author. This article has been cited by other articles in PMC. Abstract Pichia pastoris is an established protein expression host mainly applied for the production of biopharmaceuticals and industrial enzymes. This methylotrophic yeast is a distinguished production system for its growth to very high cell densities, for the available strong and tightly regulated promoters, and for the options to produce gram amounts of recombinant protein per litre of culture both intracellularly and in secretory fashion. However, not every protein of interest is produced in or secreted by P. Frequently, protein yields are clearly lower, particularly if complex proteins are expressed that are hetero-oligomers, membrane-attached or prone to proteolytic degradation.

Author:Kir Zolokree
Country:Singapore
Language:English (Spanish)
Genre:Sex
Published (Last):3 November 2012
Pages:144
PDF File Size:15.46 Mb
ePub File Size:19.60 Mb
ISBN:778-1-68905-300-5
Downloads:25910
Price:Free* [*Free Regsitration Required]
Uploader:Yozshugore



Pichia pastoris as an experimental organism Thirty years ago, Koichi Ogata first described the ability of certain yeast species to utilize methanol as a sole source of carbon and energy [1].

The methylotrophs attracted immediate attention as potential sources of single-cell protein SCP to be marketed primarily as high-protein animal feed. Unfortunately, the oil crisis of the s caused a dramatic increase in the cost of methane.

Concomitantly, the price of soybeans, the major alternative source of animal feed, fell. As a result, the economics of SCP production from methanol were never favorable. Figure 1 Open in new tab Download slide High cell density culture of P.

The centrifuge bottle on the left shows a P. Researchers at SIBIA isolated the gene and promoter for alcohol oxidase, and generated vectors, strains, and corresponding protocols for the molecular genetic manipulation of P. In , Phillips Petroleum sold its P. Biochemical studies showed that methanol utilization requires a novel metabolic pathway involving several unique enzymes [3]. The enzyme alcohol oxidase AOX catalyzes the first step in the methanol utilization pathway, the oxidation of methanol to formaldehyde and hydrogen peroxide Fig.

AOX is sequestered within the peroxisome along with catalase, which degrades hydrogen peroxide to oxygen and water. A portion of the formaldehyde generated by AOX leaves the peroxisome and is further oxidized to formate and carbon dioxide by two cytoplasmic dehydrogenases, reactions that are a source of energy for cells growing on methanol. Figure 2 The methanol pathway in P. Figure 2 Open in new tab Download slide The methanol pathway in P.

The remaining formaldehyde is assimilated to form cellular constituents by a cyclic pathway that starts with the condensation of formaldehyde with xylulose 5-monophosphate, a reaction catalyzed by a third peroxisomal enzyme dihydroxyacetone synthase DHAS.

The products of this reaction, glyceraldehyde 3-phosphate and dihydroxyacetone, leave the peroxisome and enter a cytoplasmic pathway that regenerates xylulose 5-monophosphate and, for every three cycles, one net molecule of glyceraldehyde 3-phosphate. Two of the methanol pathway enzymes, AOX and DHAS, are present at high levels in cells grown on methanol but are not detectable in cells grown on most other carbon sources e.

Expression of the AOX1 gene is controlled at the level of transcription [ 7—9 ]. Unlike GAL1 regulation, the absence of a repressing carbon source, such as glucose in the medium, does not result in substantial transcription of AOX1. The presence of methanol is essential to induce high levels of transcription [7]. As in S. Cleavage of a P. Gene replacements occur at lower frequencies than those observed in S.

Unlike homothallic strains of S. Strains with complementary markers can be mated by subjecting them to a nitrogen-limited medium. After 1 day on this medium, cells are shifted to a standard minimal medium supplemented with nutrients designed to select for complementing diploid cells not self-mated or non-mated parental cells.

The resulting diploids are stable as long as they are not subjected to nutritional stress. To obtain spore products, diploids are returned to the nitrogen-limited medium, which stimulates them to proceed through meiosis and sporulation. Spore products are handled by random spore techniques rather than micromanipulation, since P. Yet most standard classical genetic manipulations, including mutant isolation, complementation analysis, backcrossing, strain construction, and spore analysis, can be accomplished.

A variety of P. A generalized diagram of an expression vector and a list of possible vector components are shown in Fig. More detailed information on vectors and strains can be found elsewhere [ 17 , 18 ]. Table 2 shows a list of commonly used P.

Figure 3 General diagram of a P. Table 1 Relevant components of vectors used for protein expression in P.

LADISLAU DOWBOR PDF

Heterologous protein expression in the methylotrophic yeast Pichia pastoris.

.

BOGOTAZO ARTURO ALAPE PDF

.

Related Articles