HETEROLOGOUS PROTEIN EXPRESSION IN THE METHYLOTROPHIC YEAST PICHIA PASTORIS PDF

We'd like to understand how you use our websites in order to improve them. Register your interest. 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.

Author:Tashicage Tejin
Country:Reunion
Language:English (Spanish)
Genre:Music
Published (Last):7 October 2014
Pages:403
PDF File Size:7.36 Mb
ePub File Size:12.53 Mb
ISBN:684-9-21175-506-6
Downloads:72019
Price:Free* [*Free Regsitration Required]
Uploader:Gunos



Joan Lin Cereghino, James M. During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: 1 the simplicity of techniques needed for the molecular genetic manipulation of P. In this paper, we review the P. 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.

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.

The conceptual basis for the 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. 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.

There are two genes that encode alcohol oxidase in P. 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]. Techniques required for the molecular genetic manipulation of P. 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.

Expression of any foreign gene in P. 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. General diagram of a P. Relevant components of vectors used for protein expression in P. Most expression vectors have an expression cassette composed of a 0. Between the promoter and terminator sequences is a site or multiple cloning site MCS for insertion of the foreign coding sequence.

Generally, the best expression results are obtained when the first ATG of the heterologous coding sequence is inserted as close as possible to the position of the AOX1 ATG. This position coincides with the first restriction site in most MCSs. In addition, for secretion of foreign proteins, vectors are available where in-frame fusions of foreign proteins and the secretion signals of P.

Although the AOX1 promoter has been successfully used to express numerous foreign genes, there are circumstances in which this promoter may not be suitable.

For example, the use of methanol to induce gene expression may not be appropriate for the production of food products since methane, a petroleum-related compound, is one source of methanol. Also, methanol is a potential fire hazard, especially in quantities needed for large-scale fermentations. Therefore, promoters that are not induced by methanol are attractive for expression of certain genes.

Alternative promoters to the AOX1 promoter are the P. Both northern and reporter activation results indicate that the P.

GAP promoter activity levels in glycerol- and methanol-grown cells are approximately two-thirds and one-third of the level observed for glucose, respectively. The advantage of using the GAP promoter is that methanol is not required for induction, nor is it necessary to shift cultures from one carbon source to another, making strain growth more straightforward.

However, since the GAP promoter is constitutively expressed, it is not a good choice for the production of proteins that are toxic to the yeast. The FLD1 gene encodes a glutathione-dependent formaldehyde dehydrogenase, a key enzyme required for the metabolism of certain methylated amines as nitrogen sources and methanol as a carbon source [21].

The FLD1 promoter can be induced with either methanol as a sole carbon source and ammonium sulfate as a nitrogen source or methylamine as a sole nitrogen source and glucose as a carbon source. The FLD1 promoter offers the flexibility to induce high levels of expression using either methanol or methylamine, an inexpensive nontoxic nitrogen source.

There is evidence that, for certain foreign genes, the high level of expression from P AOX1 may overwhelm the post-translational machinery of the cell, causing a significant proportion of foreign protein to be misfolded, unprocessed, or mislocalized [ 22 , 23 ].

For these and other applications, moderately expressing promoters are desirable. Toward this end, the P. The PEX8 gene encodes a peroxisomal matrix protein that is essential for peroxisome biogenesis [24].

It is expressed at a low but significant level on glucose and is induced modestly when cells are shifted to methanol. The YPT1 gene encodes a GTPase involved in secretion, and its promoter provides a low but constitutive level of expression in media containing either glucose, methanol, or mannitol as carbon sources [25]. Although classical and molecular genetic techniques are generally well-developed for P.

Existing markers are limited to the biosynthetic pathway genes HIS4 from either P. The stable expression of human type III collagen illustrates the need for multiple selectable markers in P. The production of collagen requires the coexpression of prolyl 4-hydroxylase, a central enzyme in the synthesis and assembly of trimeric collagen. Recently, a new set of biosynthetic markers has been isolated and characterized: the P. Each of these selectable markers has been incorporated into expression vectors.

In addition, a series of host strains containing all possible combinations of ade1, arg4, his4 , and ura3 auxotrophies has been generated Table 2. All P. Most have one or more auxotrophic mutations which allow for selection of expression vectors containing the appropriate selectable marker gene upon transformation. Prior to transformation, all of these strains grow on complex media but require supplementation with the appropriate nutrient s for growth on minimal media.

Most P. However, two other types of host strains are available which vary with regard to their ability to utilize methanol because of deletions in one or both AOX genes. Strains with AOX mutations are sometimes better producers of foreign proteins than wild-type strains [ 30—32 ]. Since the strain must rely on the weaker AOX2 for methanol metabolism, it grows slowly on this carbon source Mut s , methanol utilization slow phenotype.

Several protease-deficient strains — SMD his4 pep4 prb1 , SMD his4 prb1 , and SMD his4 pep4 — have been shown to be effective in reducing degradation of some foreign proteins [ 23 , 33 ]. This is especially noticeable in fermenter cultures, because the combination of high cell density and lysis of a small percentage of cells results in a relatively high concentration of these vacuolar proteases.

Kex1 protease can cleave carboxy-terminal lysines and arginines. Therefore, the deletion strain was generated to inhibit carboxy-terminal proteolysis. After 40 h of fermentation, purification of intact endostatin was achieved [34]. Unfortunately, these protease-deficient cells are not as vigorous as wild-type strains with respect to PEP4. In addition to lower viability, they possess a slower growth rate and are more difficult to transform.

Therefore, the use of protease-deficient strains is only recommended in situations where other measures to reduce proteolysis have yielded unsatisfactory results.

Expression vectors are integrated into the P. This can be done in two ways. The simplest way is to restrict the vector at a unique site in either the marker gene e. The remaining transformants have undergone gene conversion events in which only the marker gene from the vector has integrated into the mutant host locus without other vector sequences.

Alternatively, certain P. This disruption of the AOX1 gene forces these strains to rely on the transcriptionally weaker AOX2 gene for growth on methanol [31] , and, as a result, these strains have a Mut s phenotype. These gene replacement strains are easily identified among transformed colonies by replica-plating them to methanol and selecting those with reduced ability to grow on methanol.

Optimization of protein expression often, but not always, includes the isolation of multicopy expression strains. A strain that contains multiple integrated copies of an expression cassette can sometimes yield more heterologous protein than single-copy strains [ 22 , 35 ]. Three approaches lead reliably to multicopy expression strains in P.

ERNESTO SNAJER PDF

Heterologous Protein Expression in the Methylotrophic Yeast Pichia Pastoris

Joan Lin Cereghino, James M. During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: 1 the simplicity of techniques needed for the molecular genetic manipulation of P. In this paper, we review the P. 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.

CAO BAKKERSBEDRIJF 2012 PDF

During the past 15 years, the methylotrophic yeast Pichia pastoris has developed into a highly successful system for the production of a variety of heterologous proteins. The increasing popularity of this particular expression system can be attributed to several factors, most importantly: 1 the simplicity of techniques needed for the molecular genetic manipulation of P. In this paper, we review the P. This site needs JavaScript to work properly. Please enable it to take advantage of the complete set of features! Clipboard, Search History, and several other advanced features are temporarily unavailable. Search: Search.

Related Articles