High-Level Secretion of Glycosylated Invertase in the Methylotrophic Yeast, Pichia Pastoris

Tschopp, Juerg; Sverlow, Genadie G.; Kosson, Ryszard; Craig, William S.; Grinna, Lynn S.

doi:10.1038/nbt1287-1305

Cited by 194 publications

(94 citation statements)

References 24 publications

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“…3). The expression yield (1.57 g/ml) of 3P was not as high as those of several heterologous proteins, such as human tumor necrosis factor (42), the antigen pertactin (p69) of Bordetella pertussis (35), and glycosylated invertase (47), which have all been expressed in P. pastoris up to several grams per liter of culture. However, the expression levels of P proteins are as high as those of two membrane proteins of influenza virus, neuraminidase (NA) and hemagglutinin (HA), which ranged from 2.5 to 3 g/ml (24) and 0.375 to 0.675 g/ml (37), respectively.…”

Section: Discussionmentioning

confidence: 97%

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

Hwang

Yamada

Honda

et al. 2000

J Virol

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Influenza virus RNA polymerase with the subunit composition PB1-PB2-PA is a multifunctional enzyme with the activities of both synthesis and cleavage of RNA and is involved in both transcription and replication of the viral genome. In order to produce large amounts of the functional viral RNA polymerase sufficient for analysis of its structure-function relationships, the cDNAs for RNA segments 1, 2, and 3 of influenza virus A/PR/8, each under independent control of the alcohol oxidase gene promoter, were integrated into the chromosome of the methylotrophic yeast Pichia pastoris. Simultaneous expression of all three P proteins in the yeast P. pastoris was achieved by the addition of methanol. To purify the P protein complexes, a sequence coding for a histidine tag was added to the PB2 protein gene at its N terminus. Starting from the induced P. pastoris cell lysate, we partially purified a 3P complex by Ni 2؉ -agarose affinity column chromatography. The 3P complex showed influenza virus model RNA-directed and ApG-primed RNA synthesis in vitro but was virtually inactive without addition of template or primer. The kinetic properties of model template-directed RNA synthesis and the requirements for template sequence were analyzed using the 3P complex. Furthermore, the 3P complex showed capped RNA-primed RNA synthesis. Thus, we conclude that functional influenza virus RNA polymerase with the catalytic properties of a transcriptase is formed in the methylotrophic yeast P. pastoris. Influenza A virus contains eight different RNA segments of negative polarity in its genome, each encoding one or two unique viral proteins. The viral RNA polymerase is associated with each RNA segment as a viral component and in infected cells, responsible for both transcription and replication of the viral genome (for reviews, see references 7, 13, and 21). The pathway for the synthesis of viral mRNA involves multiple-step reactions, consisting of endonucleolytic cleavage of host cell mRNA, capped oligonucleotide-primed transcription of viral RNA (vRNA), and the addition of a poly(A) tail to the nascent mRNA. On the other hand, replication takes place in two steps, vRNA-directed synthesis of full-length complementary RNA (cRNA) without any modification at both the 5Ј and 3Ј termini and cRNA-directed reproduction of vRNA.Starting from the viral ribonucleoprotein (RNP), we isolated an RNA-3P (PB1, PB2, and PA) protein complex without NP by equilibrium centrifugation in cesium sulfate or cesium chloride (15, 16). The RNA-3P complex is enzymatically active in the in vitro synthesis of short attenuated RNA chains (but NP is needed for RNA chain elongation [12]), indicating that the three P proteins participate in the catalytic function of RNA polymerization. The vRNA-free 3P complex, consisting of one molecule each of the three P proteins, was isolated from the RNA-3P complex by centrifugation in cesium chloride or cesium trifluoroacetate (11). The 3P complexes exhibited RNA synthesis only when a model vRNA template with 5Ј-and 3Ј-terminal cons...

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Section: Discussionmentioning

confidence: 97%

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

Hwang

Yamada

Honda

et al. 2000

J Virol

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“…of the methylotrophic yeast P. pastoris has been used for the characterization of genes encoding sugarmetabolizing enzymes such as invertase 34) and 6-SFT. 33) Our results using this system indicate that both Wft1 and Wft2 encode fructan-synthesizing enzymes.…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of Sucrose:Sucrose 1-Fructosyltransferase and Sucrose:Fructan 6-Fructosyltransferase Associated with Fructan Accumulation in Winter Wheat during Cold Hardening

Kawakami

Yoshida

2002

Bioscience, Biotechnology, and Biochemistry

112

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“…The methylotrophic yeast P. pastorishas been reported previously to produce high levels of heterologous proteins (10) including human serum albumin (2), invertase (26), mouse epidermal growth factor (9) and aprotinin (27). One of the advantages of this eukaryotic expression system is the ability to secrete high levels of recombinant protein into a protein-free growth medium, facilitating protein purification.…”

Section: Introductionmentioning

confidence: 99%

Recombinant HMG1 Protein Produced in Pichia pastoris : A Nonviral Gene Delivery Agent

Mistry¹,

Falciola²,

Monaco³

et al. 1997

BioTechniques

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High-Level Secretion of Glycosylated Invertase in the Methylotrophic Yeast, Pichia Pastoris

Cited by 194 publications

References 24 publications

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

Expression of Functional Influenza Virus RNA Polymerase in the Methylotrophic Yeast Pichia pastoris

Molecular Characterization of Sucrose:Sucrose 1-Fructosyltransferase and Sucrose:Fructan 6-Fructosyltransferase Associated with Fructan Accumulation in Winter Wheat during Cold Hardening

Recombinant HMG1 Protein Produced in Pichia pastoris : A Nonviral Gene Delivery Agent

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