Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone

Vad, Randi; Nafstad, Eidi; Dahl, Linn Anita; Gabrielsen, Odd S.

doi:10.1016/j.jbiotec.2004.12.004

Cited by 50 publications

(30 citation statements)

References 42 publications

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“…This single cell organism is capable of many of the functions carried out in higher eukaryotic cells such as glycosylation, folding and disulphide bond formation, and yet its molecular manipulation needs simple and established techniques used for prokaryotes (Bollok et al 2009). This system is less expensive than insect and mammalian tissue culture cell systems and has been widely used, both intracellulary and extracellulary, for the production of negligible to high quantities of foreign heterologous proteins (Macauley-Patrick et al 2005;Vad et al 2005).…”

Section: Discussionmentioning

confidence: 99%

Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastorisan alpha 1-antitrypsin in Pichia pastoris

Arjmand¹,

Lotfi²,

Shamsara³

et al. 2013

Electron. J. Biotechnol.

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Background: Human alpha 1-antitrypsin (AAT) is a potent inhibitor of multiple serine proteases, and protects tissues against their harmful effects. Individuals with reduced or abnormal production of this inhibitor need intravenous administration of exogenous protein. In this study, we employed the methylotrophic (methanol utilizing) yeast Pichia pastoris (P. pastoris) as a preferential host for efficient production and secretion of recombinant AAT. Furthermore, we examined different strategies to maximize the yield of the secreted protein.Results: Our findings revealed that optimizing the codon usage of AAT gene for P. pastoris had positive effects on the level of secreted AAT under the control of inducible alcohol oxidase 1 (AOX1) and constitutive glycerol aldehyde phosphate dehydrogenase (GAP) promoters. Compared to AOX1, the GAP promoter increased the yield of AAT by more than two fold. It was also demonstrated that the human AAT native signal sequence was more effective than the well-known yeast signal sequence, alpha mating factor (α-MF). Doubling gene dosage nearly doubled the production of AAT, though dosages exceeding this limit had negative effects on the yield. Conclusion: P. pastoris is shown to be an efficient expression system for production of recombinant and biologically active AAT. Also different strategies could be used to elevate the amount of this secretable protein.

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Section: Discussionmentioning

confidence: 99%

Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastorisan alpha 1-antitrypsin in Pichia pastoris

Arjmand¹,

Lotfi²,

Shamsara³

et al. 2013

Electron. J. Biotechnol.

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“…However its presence in the extracellular environment has already been reported. In particular, Kieffer et al (20) observed that bovine chromaffin cells secrete ubiquitin upon nicotinic stimulation and Vad et al (45) showed that a Pichia pastoris strain engineered to produce human parathyroid hormone cosecreted ubiquitin in the culture medium.…”

Section: Discussionmentioning

confidence: 99%

Pichia anomalaDBVPG 3003 Secretes a Ubiquitin-Like Protein That Has Antimicrobial Activity

Ingeniis

Raffaelli

Ciani

et al. 2009

Appl Environ Microbiol

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The yeast strain Pichia anomala DBVPG 3003 secretes a killer toxin (Pikt) that has antifungal activity against Brettanomyces/Dekkera sp. yeasts. Pikt interacts with ␤-1,6-glucan, consistent with binding to the cell wall of sensitive targets. In contrast to that of toxin K1, secreted by Saccharomyces cerevisiae, Pikt killer activity is not mediated by an increase in membrane permeability. Purification of the toxin yielded a homogeneous protein of about 8 kDa, which showed a marked similarity to ubiquitin in terms of molecular mass and N-terminal sequences. Pikt is also specifically recognized by anti-bovine ubiquitin antibodies and, similar to ubiquitin-like peptides, is not absorbed by DEAE-cellulose. However, Pikt differs from ubiquitin in its sensitivity to proteolytic enzymes. Therefore, Pikt appears to be a novel ubiquitin-like peptide that has killer activity.After its initial discovery in Saccharomyces cerevisiae (2), the killer phenotype has been found in yeasts belonging to the genera Candida, Cryptococcus, Debaryomyces, Hanseniaspora, Hansenula, Kloeckera, Kluyveromyces, Metschnikowia, Pichia, Rhodotorula, Schwanniomyces, Torulopsis, Trichosporon, Williopsis, and Zygosaccharomyces (9,16,19,25,28,53,56). The distribution of this character among natural yeast isolates and in laboratory strains is accompanied by killer toxins with large biodiversity, in terms of their biochemical characteristics, genetic determinants, and spectra of action (25). In spite of this diversity, all known killer toxins are proteins (1, 31) or glycoproteins (5,11,28,43,54) that kill sensitive cells via a two-step mode of action. During the first step, the killer toxin binds a receptor site on the cell wall of its sensitive target. In the second step, which has been far less characterized, the killer toxin is assumed to interact with receptors on the cell membrane and to kill the sensitive cells via different mechanisms. These include cell membrane permeabilization, cell cycle perturbation, inhibition of DNA synthesis, and inhibition of ␤-1,3-glucan synthetase activity (7,14,15,41,49,53).As the spectrum of action of some toxins has extended to microbial pathogens of clinical interest, killer toxins and/or killer toxin-like antibodies and mimotopes are of great relevance to medicine (8,25). Other toxins that exert a killing action on spoilage yeasts have interesting applications in the fermentative (46) and food and feed industries (10,11,12,23,32,44), where they can be used as "natural" food antimicrobials.In a previous study, we showed that Pichia anomala DBVPG 3003 produces a killer toxin (known as Pikt) that is active against Dekkera/Brettanomyces sp. yeasts (12). These yeasts can develop in white and red wines, resulting in unpleasant odors and tastes (18). Thus, they represent a major problem in the wine industry. Pikt is stable at acidic pH and in a range of temperatures between 20°C and 40°C (12), as are other toxins produced by different strains of P. anomala (25,26,37). Moreover, Pikt maintains its killing activity for ...

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“…For example, human parathyroid hormone (hPTH), a promising agent in the treatment of osteoporosis, does not contain any cystein residues. Its expressed amount can be considerably enhanced by the co-expression of protein disulfide isomerase from S. cerevisiae (ScPDI) [42]. This may be indirect, or may be caused by the chaperone activity of PDI.…”

Section: Endoplasmic Reticulum Resident Proteins and Their Effect On mentioning

confidence: 99%

Improving the Secretory Production of the Heterologous Protein in Pichia pastoris by Focusing on Protein Folding

Zhu

Chen

et al. 2014

Appl Biochem Biotechnol

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Pichia pastoris has currently been developed as an effective host system for the expression of heterologous genes owing to its potential use for the production of soluble and high-yield proteins. However, the secretory production of the different heterologous proteins in P. pastoris varies widely. Some factors restrict the effective secretory production of heterologous proteins in P. pastoris, among which the folding and processing of proteins is a major one. Besides optimizing the fermentative process, current strategies focus on investigating protein folding process. Thus, this paper is the first time to review the improvement of the secretory production of the heterologous protein in P. pastoris by focusing on its folding process.

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Engineering of a Pichia pastoris expression system for secretion of high amounts of intact human parathyroid hormone

Cited by 50 publications

References 42 publications

Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastorisan alpha 1-antitrypsin in Pichia pastoris

Elevating the expression level of biologically active recombinant human alpha 1-antitrypsin in Pichia pastorisan alpha 1-antitrypsin in Pichia pastoris

Pichia anomalaDBVPG 3003 Secretes a Ubiquitin-Like Protein That Has Antimicrobial Activity

Improving the Secretory Production of the Heterologous Protein in Pichia pastoris by Focusing on Protein Folding

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