Disruption of the KEX1 gene inPichia pastoris allows expression of full-length murine and human endostatin

Boehm, Thomas; Pirie-Shepherd, Steven; Trinh, Loc-Ba; Shiloach, Joseph; Folkman, Judah

doi:10.1002/(sici)1097-0061(199905)15:7<563::aid-yea398>3.0.co;2-r

Cited by 60 publications

(21 citation statements)

References 18 publications

(18 reference statements)

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“…Thus, the N-terminal signal sequence in K28 pptox functions equally effectively in all four yeast genera, indicating that it might have the potential for being used as a novel and unique signal sequence suitable for foreign protein secretion. Furthermore, this is the first time that a biologically active virus toxin has been successfully expressed and secreted in C. glabrata and P. pastoris, confirming more recent reports on the existence of Kex2p/Kex1p homologous processing machinery that is required for the in vivo processing and maturation of protein precursors (1,2).…”

Section: Resultssupporting

confidence: 84%

Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein byCandida glabrata,Pichia pastoris,Saccharomyces cerevisiae, andSchizosaccharomyces pombe

Eiden-Plach

Zagorc

Heintel

et al. 2004

Appl Environ Microbiol

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Besides its importance as model organism in eukaryotic cell biology, yeast species have also developed into an attractive host for the expression, processing, and secretion of recombinant proteins. Here we investigated foreign protein secretion in four distantly related yeasts (Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe) by using green fluorescent protein (GFP) as a reporter and a viral secretion signal sequence derived from the K28 preprotoxin (pptox), the precursor of the yeast K28 virus toxin. In vivo expression of GFP fused to the N-terminal pptox leader sequence and/or expression of the entire pptox gene was driven either from constitutive (PGK1 and TPI1) or from inducible and/or repressible (GAL1, AOX1, and NMT1) yeast promoters. In each case, GFP entered the secretory pathway of the corresponding host cell; confocal fluorescence microscopy as well as sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of cell-free culture supernatants confirmed that GFP was efficiently secreted into the culture medium. In addition to the results seen with GFP, the full-length viral pptox was correctly processed in all four yeast genera, leading to the secretion of a biologically active virus toxin. Taken together, our data indicate that the viral K28 pptox signal sequence has the potential for being used as a unique tool in recombinant protein production to ensure efficient protein secretion in yeast.Over the years, heterologous protein expression in yeast species such as Candida glabrata, Pichia pastoris, Saccharomyces cerevisiae, and Schizosaccharomyces pombe has become an important tool in the production of therapeutic and commercially relevant proteins. The advantage of a yeast-based expression system is due to the fact that as a eukaryotic microorganism, yeast combines ease of genetic manipulation and cell growth with a well-known capacity to perform complex posttranslational protein modifications (4,22,29). Because yeast per se secretes only low levels of endogenous proteins, a secreted recombinant protein usually contributes significantly to the total amount of proteins in the culture medium. Thus, directing a foreign protein for secretion is an important initial step in its subsequent purification.While the vast majority of heterologous proteins has been expressed within the cytosol of the corresponding host, only a few proteins have been successfully secreted into the extracellular medium. In most eukaryotic proteins, the critical initial step in protein secretion is their co-and/or posttranslational translocation into the lumen of the endoplasmic reticulum (ER) followed by subsequent sorting into the Golgi network. Foreign protein import into the ER is usually achieved by fusing the protein of interest in frame to a homologous secretion signal sequence derived from a naturally secreted protein of the corresponding host, thus conferring secretion competence to the desired protein fusion (13,23).In yeast, the most widely used secretion sign...

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Section: Resultssupporting

confidence: 84%

Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein byCandida glabrata,Pichia pastoris,Saccharomyces cerevisiae, andSchizosaccharomyces pombe

Eiden-Plach

Zagorc

Heintel

et al. 2004

Appl Environ Microbiol

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“…Despite intensive efforts, large quantities of recombinant protein sufficient for clinical trials were not available until recently (6). The difficulties in protein production, long-term storage of bioactive protein, and the cumbersome daily administration may be overcome through transfer of the endostatin gene.…”

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confidence: 99%

Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases

Sauter

Martinet

Zhang

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

230

162

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Inhibition of angiogenesis has been shown to be an effective strategy in cancer therapy in mice. However, its widespread application has been hampered by difficulties in the large-scale production of the antiangiogenic proteins. This limitation may be resolved by in vivo delivery and expression of the antiangiogenic genes. We have constructed a recombinant adenovirus that expresses murine endostatin that is biologically active both in vitro, as determined in endothelial cell proliferation assays, and in vivo, by suppression of angiogenesis induced by vascular endothelial growth factor 165. Persistent high serum levels of endostatin (605-1740 ng͞ml; mean, 936 ng͞ml) were achieved after systemic administration of the vector to nude mice, which resulted in significant reduction of the growth rates and the volumes of JC breast carcinoma and Lewis lung carcinoma (P < 0.001 and P < 0.05, respectively). In addition, the endostatin vector treatment completely prevented the formation of pulmonary micrometastases in Lewis lung carcinoma (P ‫؍‬ 0.0001). Immunohistochemical staining of the tumors demonstrated a decreased number of blood vessels in the treatment group versus the controls. In conclusion, the present study clearly demonstrates the potential of vectormediated antiangiogenic gene therapy as a component in cancer therapy.antiangiogenesis ͉ cancer ͉ gene therapy

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“…When Hirudin was expressed in P. pastoris it was found that the molecule had degraded products where up to three amino acids were clipped from the C-terminus [25]. When human and murine endostatin were expressed in P. pastoris the C-terminal lysine was clipped [13]. In both the cases, inactivation of the KEX1 gene was helpful to express the full length proteins.…”

Section: Discussionmentioning

confidence: 99%

“…Aspergillus oryzae also has an ortholog of Kex1 termed KexA and displays a serine-type carboxypetidase activity and a broader substrate [12]. Amino acid comparison of P. pastoris and S. cerevisiae Kex1protein shows only 36% identity and 43.7% similarity [13]. Nevertheless, amino acid residues involved in the catalytic triad of S. cerevisiae (Ser198, Asp406 and His470) are conserved [14].…”

Section: Introductionmentioning

confidence: 99%

Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris

Sreenivas

Krishnaiah

Mohan

et al. 2016

Protein Expression and Purification

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a b s t r a c tInsulin glargine is a slow acting analog of insulin used in diabetes therapy. It is produced by recombinant DNA technology in different hosts namely E. coli and Pichia pastoris. In our previous study, we have described the secretion of fully folded two-chain Insulin glargine into the medium by over-expression of Kex2 protease. The enhanced levels of the Kex2 protease was responsible for the processing of the glargine precursor with in the host. Apart from the two-chain glargine product we observed a small proportion of arginine clipped species. This might be due to the clipping of arginine present at the Cterminus of the B-chain as it is exposed upon Kex2 cleavage. The carboxypeptidase precursor Kex1 is known to be responsible for clipping of C-terminal lysine or arginine of the proteins or peptides. In order to address this issue we created a Kex1 knock out in the host using Cre/loxP mechanism of targeted gene deletion. When two-chain glargine was expressed in the Kex1 knock out host of P. pastoris GS115 the Cterminal clipped species reduced by~80%. This modification further improved the process by reducing the levels of product related impurities.

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Disruption of the KEX1 gene inPichia pastoris allows expression of full-length murine and human endostatin

Cited by 60 publications

References 18 publications

Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein byCandida glabrata,Pichia pastoris,Saccharomyces cerevisiae, andSchizosaccharomyces pombe

Viral Preprotoxin Signal Sequence Allows Efficient Secretion of Green Fluorescent Protein byCandida glabrata,Pichia pastoris,Saccharomyces cerevisiae, andSchizosaccharomyces pombe

Adenovirus-mediated gene transfer of endostatin in vivo results in high level of transgene expression and inhibition of tumor growth and metastases

Disruption of KEX1 gene reduces the proteolytic degradation of secreted two-chain Insulin glargine in Pichia pastoris

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