Low-Temperature Increases the Yield of Biologically Active Herring Antifreeze Protein in Pichia pastoris

Li, Zhengjun; Xiong, Fei; Lin, Qingsong; d’Anjou, Marc; Daugulis, Andrew J.; Yang, Dan; Hew, Choy L.

doi:10.1006/prep.2001.1395

Cited by 177 publications

(123 citation statements)

References 26 publications

(6 reference statements)

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“…If so, this activity was considered all or nothing in that the recombinant protein was either susceptible to endogenous proteases or it was not. Should this condition exist, the use of protease inhibitors or protease-defi cient strains might alleviate the problem, and extended production phases are again favored (Gleeson et al 1998;Li et al 2001). Yet, the yield profi le of rOvIFN-τ did not fi t these existing precepts.…”

Section: Discussionmentioning

confidence: 99%

Pichia pastoris fermentation optimization: energy state and testing a growth-associated model

Plantz

Sinha

Villarete³

et al. 2006

Appl Microbiol Biotechnol

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Section: Discussionmentioning

confidence: 99%

Pichia pastoris fermentation optimization: energy state and testing a growth-associated model

Plantz

Sinha

Villarete³

et al. 2006

Appl Microbiol Biotechnol

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“…However, since the protease-normal strain had a slow methanol utilization phenotpye (Mut s ), it can not be concluded that the protease-deficient strain performed any better with regard to recombinant protein and biomass production levels than if the comparison had been made with a methanol plus phenotype (Mut + ) strain. Comparison of protease-deficient and wild-type strains for the production of herring antifreeze protein demonstrated that, while the wild-type strain began product secretion a day earlier than the proteasedeficient strain, a higher yield of intact product was obtained after purification using the protease-deficient strain (12.8% as opposed to 4% from the wild-type) [64]. This was probably due to the lack of proteolytic degradation of the product using the protease-deficient strain.…”

Section: Cell-level Strategiesmentioning

confidence: 99%

“…Li and co-workers [64] have shown that lowering the process temperature (from 30 • C to 23 • C) increased the yield of herring antifreeze proteins from 5.3 mg/l to 18.0 mg/l, and also increased cell viability. Hong et al [48] achieved higher laccase activity by decreasing the cultivation temperature (from 30 • C to 20 • C) and by reducing methanol concentration (from 1.0% to 0.5%).…”

Section: Cultivation-level Strategiesmentioning

confidence: 99%

Heterologous protein production using the Pichia pastoris expression system

Macauley-Patrick

Fazenda

McNeil

et al. 2005

Yeast

1,096

633

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The Pichia pastoris expression system is being used successfully for the production of various recombinant heterologous proteins. Recent developments with respect to the Pichia expression system have had an impact on not only the expression levels that can be achieved, but also the bioactivity of various heterologous proteins. We review here some of these recent developments, as well as strategies for reducing proteolytic degradation of the expressed recombinant protein at cultivation, cellular and protein levels. The problems associated with post-translational modifications performed on recombinant proteins by P. pastoris are discussed, including the effects on bioactivity and function of these proteins, and some engineering strategies for minimizing unwanted glycosylations. We pay particular attention to the importance of optimizing the physicochemical environment for efficient and maximal recombinant protein production in bioreactors and the role of process control in optimizing protein production is reviewed. Finally, future aspects of the use of the P. pastoris expression system are discussed with regard to the production of complex membrane proteins, such as G protein-coupled receptors, and the industrial and clinical importance of these proteins.

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“…Protein concentrations were determined by Bradford assay as described in standard protocol. 19 An amount of 40 mg total protein from whole cell lysate was resolved on 7.5, 10 and 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA). The membranes were blocked with 5% nonfat milk Tris-buffered saline Tween-20 buffer 1 h at room temperature, and incubated for 2 h with primary antibodies.…”

Section: Western Blottingmentioning

confidence: 99%

Suppression of N-Ras by shRNA-expressing plasmid increases sensitivity of HepG2 cells to vincristine-induced growth inhibition

et al. 2009

Cancer Gene Ther

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Oncogenic ras genes relate to the development of human cancers. In this study, we used a plasmid-mediated short-hairpin RNA (shRNA) targeting N-ras gene to combine with clinical drug vincristine (VCR) for the treatment of human hepatoma cells. Our results showed that anti-N-Ras shRNA expression vector (pCSH1-shNR) knocked down the target mRNA and protein. Higher efficacy on growth inhibition was observed when pCSH1-shNR was combined with VCR. This synergistic effect was associated with abrogation of VCR-induced overexpressions of P-glycoprotein and multidrug resistance-associated protein 1 by pCSH1-shNR through downregulations of N-Ras and total Ras. Western blot analysis showed that pCSH1-shNR-induced downregulations of N-Ras and total Ras were potentiated by VCR. Following Ras downregulation, phosphorylations of ERK1/2 and Akt were dramatically inhibited by combinatory treatment. The data showed that pCSH1-shNR-induced inhibition of nuclear factor-kB was enhanced by VCR. In addition, the combination of pCSH1-shNR and VCR synergistically inhibited the growth of human hepatoma HepG2 in vivo. Our findings suggested that combination of gene-specific therapeutics and appropriate chemotherapeutic agents might be a promising approach for the treatment of human hepatocellular carcinoma.

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Low-Temperature Increases the Yield of Biologically Active Herring Antifreeze Protein in Pichia pastoris

Cited by 177 publications

References 26 publications

Pichia pastoris fermentation optimization: energy state and testing a growth-associated model

Pichia pastoris fermentation optimization: energy state and testing a growth-associated model

Heterologous protein production using the Pichia pastoris expression system

Suppression of N-Ras by shRNA-expressing plasmid increases sensitivity of HepG2 cells to vincristine-induced growth inhibition

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