Secretion of a Variant of Human Single‐Chain Urokinase‐Type Plasminogen Activator without an N‐Glycosylation Site in the Methylotrophic Yeast, Pichia pastoris and Characterization of the Secreted Product

TSUJIKAWA, MUNEO; OKABAYASHI, KEN; MORITA, MASANORI; Tanabe, Toshizumi

doi:10.1002/(sici)1097-0061(199605)12:6<541::aid-yea935>3.3.co;2-1

Cited by 10 publications

(13 citation statements)

References 35 publications

(46 reference statements)

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“…AOX1-deleted transformants could grow slowly in the methanol medium because they had another alcohol oxidase encoded by the AOX2 gene, which is expressed more weakly than the AOX1 gene . The frequency of AOX1 disruption by double crossing-over (37%) was identical to the frequency reported for the production of hepatitis B antigen by Cregg et al (1987), tumour necrosis factor by Sreekrishna et al (1989) and human single-chain urokinase-type plasminogen activator (Tsujikawa et al, 1996).…”

supporting

confidence: 77%

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

Céline¹,

Chemardin²,

Bigey³

et al. 2004

Yeast

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Ovine leptin was cloned in the methylotrophic yeast Pichia pastoris using a pPIC9K vector. Leptin was produced and secreted into the culture medium using the Saccharomyces cerevisiae α-mating factor prepro signal by five clones. Expression levels of leptin varied from clone to clone, depending on the copy number of the ob gene. Highest expression was observed with the single-copy clone S27 (250 mg/l). The modifications of culture conditions in batch and fed-batch culture increase the yield of protein. The use of higher cell concentration (63 g/l) before induction of oLept associate with a regulation of pH at 3.2, which decreases the effects of proteolysis, increases the expression level of the oLept to 402 mg/l. Moreover, compared with the non-producer clone, we observed a drastic decrease in growth rate and biomass yield in the leptin-producing clones. At the end of the fed-batch phase at pH 3.2 with clone S27, mortality rate reached 17.3%. Results showed that recombinant leptin production induced metabolic stress, and a negative impact on biomass yield and growth rate. We characterized the recombinant leptin produced by clone S27. It exhibited a molecular mass of 16 kDa, an N-terminal amino acid sequence identical to that of ovine leptin but with an additional tyrosine introduced by the cloning site. Moreover, it was found to be biologically active in vitro. The available production of a large quantity of oLept will strengthen the functional study for theorical and practical purposes.

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supporting

confidence: 77%

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

Céline¹,

Chemardin²,

Bigey³

et al. 2004

Yeast

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“…Because the pmr1‐ Δ mutation caused a defect of vacuolar protein sorting (see above), which could lead to an increased proteolytic activation of uPA (Agaphonov et al , 2005a), uPA‐Q 302 secreted by the pmr‐ Δ mutant and wild‐type control strain was analyzed by Western blotting. It was previously observed that buffering of the culture medium with ammonium phosphate decreased the proteolysis of uPA (Tsujikawa et al , 1996; Agaphonov et al , 2005a). Indeed, the major band of uPA‐Q 302 from the culture supernatants migrated in SDS‐PAGE as a ∼45‐kDa protein (the calculated molecular weight of uPA‐Q 302 is 46 kDa).…”

Section: Resultsmentioning

confidence: 99%

Inactivation of theHansenula polymorpha PMR1gene affects cell viability and functioning of the secretory pathway

Agaphonov¹,

Plotnikova²,

Fokina³

et al. 2007

FEMS Yeast Research

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In yeast, functions of the endoplasmic reticulum (ER) depend on the Golgi apparatus Ca2+ pool, which is replenished by the medial-Golgi ion pump Pmr1p. Here, to dissect the role of the Golgi Ca2+ pool in protein folding and elimination of unfolded proteins in the ER, the manifestations of the pmr1 mutation in yeast Hansenula polymorpha were studied. The PMR1 gene was disrupted in a H. polymorpha diploid strain. Haploid segregants of this diploid bearing the disruption allele were viable, though they showed a severe growth defect on synthetic medium and rapidly died during storage at low temperature. Disruption of H. polymorpha PMR1 led to defects of the Golgi-hosted protein glycosylation and vacuolar protein sorting. This mutation increased the survival rate of H. polymorpha cells upon treatment with the proapoptotic drug amiodarone. Unlike Saccharomyces cerevisiae, the H. polymorpha pmr1 mutant was not hypersensitive to chemicals that induce the accumulation of unfolded proteins in the ER, indicating that the elimination of unfolded proteins from the ER was not essentially affected. At the same time, the pmr1 mutation improved the secretion of human urokinase and decreased its intracellular aggregation, indicating an influence of the mutation on the protein folding in the ER.

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“…Although there is little information concerning the mechanism and speci¢city of O-glycosylation in P. pastoris, the presence of O-glycosylation has been reported in some heterologous proteins, such as the Aspergillus awamori glucoamylase catalytic domain [47], human IGF-1 [23], barley K-amylases 1 and 2 [48], and human single-chain urokinase-type plasminogen activator [49].…”

Section: O-linked Glycosylationmentioning

confidence: 99%

Heterologous protein expression in the methylotrophic yeast Pichia pastoris

Cereghino

2000

FEMS Microbiology Reviews

601

853

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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. pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology; (2) the ability of P. pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing; and (4) the availability of the expression system as a commercially available kit. In this paper, we review the P. pastoris expression system: how it was developed, how it works, and what proteins have been produced. We also describe new promoters and auxotrophic marker/host strain combinations which extend the usefulness of the system.

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Secretion of a Variant of Human Single‐Chain Urokinase‐Type Plasminogen Activator without an N‐Glycosylation Site in the Methylotrophic Yeast, Pichia pastoris and Characterization of the Secreted Product

Cited by 10 publications

References 35 publications

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone

Inactivation of theHansenula polymorpha PMR1gene affects cell viability and functioning of the secretory pathway

Heterologous protein expression in the methylotrophic yeast Pichia pastoris

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