High level expression of proinsulin in the yeast, Saccharomyces cerevisiae

Cousens, Lawrence S.; Shuster, Jeffrey R.; Gallegos, C; Ku, Lailing; Stempien, Michelle M.; Urdea, Mickey S.; Sánchez-Pescador, Ray; Taylor, Alice Y.; Tekamp-Olson, Patricia

doi:10.1016/0378-1119(87)90190-9

Cited by 78 publications

(35 citation statements)

References 25 publications

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“…The latter medium allows the selection of yeast cells containing a high plasmid copy number since the plasmid leu2-d is poorly expressed. The expression of recombinant IN was under the control of the alcohol dehydrogenase 2/glyceraldehyde-3-phosphate dehydrogenase-inducible hybrid promoter (11 on a YCAD selective medium, precultured in YNB liquid medium, and then incubated in YPDA complete medium for 3 days. Cells were harvested for 10 min at 4,000 ϫ g and broken with glass beads in 5 mM HEPES (pH 7.6)-5 mM EDTA-1 mM DTT.…”

Section: Abbreviationsmentioning

confidence: 99%

Functional Interactions of Human Immunodeficiency Virus Type 1 Integrase with Human and Yeast HSP60

Parissi

Calmels

Soultrait

et al. 2001

J Virol

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The HIV-1 integrase IN catalyzes a critical step in the infectious cycle of this retrovirus. The mechanism leading to the integration of the proviral DNA into the host genome can be divided into two separate reactions (1). HIV-1 IN catalyzes first a hydrolytic reaction of the proviral DNA substrate, termed "processing," and second a transesterification step termed "joining" or "strand transfer," which allows the insertion of the provirus into the nuclear genome. In the processing reaction where OH Ϫ is the nucleophilic agent, IN is able to endonucleolytically remove in vitro two nucleotides from an oligonucleotide (ODN) mimicking the retroviral LTR ends of HIV-1. In the joining or strand transfer reaction, by using the processed LTR as a nucleophile, IN catalyzes a transesterification hydrolytic reaction. The resulting double-stranded proviral DNA is part of a large nucleoprotein structure termed the PIC. This complex contains the information necessary for nuclear localization and the enzymatic machinery required to insert the proviral DNA into the host genome (for a review of the PIC, see reference 4). In addition to IN and the proviral DNA, the PIC has been reported to contain various viral and cellular proteins. However, a precise description of the viral and cellular partners involved in this complex is not yet well defined.Although recombinant IN purified from Saccharomyces cerevisiae or bacteria can perform all the in vitro reactions using synthetic substrates (9), there is much evidence that proteins present in cytoplasmic extracts from uninfected cells are also involved in the integration process. Two proteins, the barrierto-autointegration factor and HMG I(Y), have been identified as specific cofactors (8,17). In addition, Kalpana et al. (21), using the yeast two-hybrid system, reported the isolation of a host factor, the integrase-interacting protein 1 (Ini1), as a binding partner of HIV-1 IN. Ini1 displays a high degree of sequence similarity to the yeast protein SNF5, a factor involved in the transcriptional activation of a number of genes.We have previously shown that the expression of HIV-1 IN in yeast induces a lethal phenotype (5), while the expression of an inactive mutated IN does not. These results strongly suggest that the lethal phenotype could be due to cell death by DNA damage induced by the IN activity. Moreover, the inactivation of the SNF5 transcription factor gene abolished the lethal phenotype induced by the expression of HIV-1 IN in yeast, indicating that SNF5 is able to interact with . Therefore, the use of the yeast system should facilitate the identification of IN-interacting proteins, thus allowing further studies in a cellular context by exploring the effect of gene inactivation on the IN-induced lethal effect.To identify yeast cellular proteins in addition to SNF5, whose human counterparts might participate in retroviral DNA integration, we developed an in vitro system to detect the interactions between HIV-1 IN and S. cerevisiae proteins. Using IN-affinity chromatography, we i...

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Section: Abbreviationsmentioning

confidence: 99%

Functional Interactions of Human Immunodeficiency Virus Type 1 Integrase with Human and Yeast HSP60

Parissi

Calmels

Soultrait

et al. 2001

J Virol

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“…The C22 (119 aa), el (130 aa), ns5 (942 aa), and chimeric C25 (858 aa) antigens (see Fig. 1 for relative location) were expressed as internal antigens within the yeast Saccharomyces cerevisiae as C-terminal fusions with human superoxide dismutase (SOD) by using methods described previously for the generation of the C100-3 (363 aa) antigen (5,17). The C33C antigen (266 aa; Fig.…”

Section: Methodsmentioning

confidence: 99%

Diagnosis of hepatitis C virus (HCV) infection using an immunodominant chimeric polyprotein to capture circulating antibodies: reevaluation of the role of HCV in liver disease.

Chien¹,

Choo²,

Tabrizi³

et al. 1992

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

108

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Structural and nonstructural regions of the HCV-encoded polyprotein have been expressed in recombinant yeast, bacteria, or insect cells and used to capture and measure reactive antibodies circulating in different individuals. The putative nucleocapsid protein (C) and nonstructural proteins 3-5 (NS3-NS5) were found to contain the most immunodominant epitopes. The NS3, NS4, and C regions were expressed in yeast in the form of a fused, chimeric polyprotein (C25) and a capture assay for reactive antibody was developed. This anti-C25 assay detects all previously identified HCV-seropositive cases and provides a substantially more sensitive diagnostic for both acute and chronic HCV infections than the current anti-C100-3 (NS4) assay. Anti-C25 was detected more frequently than anti-C100-3 in chronic, transfusion-associated non-A, non-B hepatitis patients from the United States (95% vs. 71%) and Japan (98% vs. 82%), in cryptogenic cirrhosis patients from the United States (62% vs. 28%), and in hepatitis B surface antigen-negative cases of hepatocellular carcinoma from Japan (83% vs. 63%). These data indicate that HCV has a greater role in these liver diseases than was previously thought. In volunteer United States blood donors sampled following the introduction of anti-C100-3 screening, the prevalence of anti-C25 and anti-C100-3 was 0.5% and 0.08%, respectively.

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“…Более того, за счет слияния ее кДНК с другим клонируемым геном удавалось увеличить выход интере-сующего гетерологичного белка. В виде таких гибридных белков в дрожжах были успешно синтезированы проин-сулин человека (Cousens et al, 1987) и белки оболочки вируса иммунодефицита (HIV) (Barr et al, 1987).…”

Section: синтез гетерологичных белковunclassified

Genetically modified microorganisms as producers of biologically active compounds

Падкина

Самбук

2015

Ecological genetics

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High level expression of proinsulin in the yeast, Saccharomyces cerevisiae

Cited by 78 publications

References 25 publications

Functional Interactions of Human Immunodeficiency Virus Type 1 Integrase with Human and Yeast HSP60

Functional Interactions of Human Immunodeficiency Virus Type 1 Integrase with Human and Yeast HSP60

Diagnosis of hepatitis C virus (HCV) infection using an immunodominant chimeric polyprotein to capture circulating antibodies: reevaluation of the role of HCV in liver disease.

Genetically modified microorganisms as producers of biologically active compounds

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