Production of specific antibodies against protein A fusion proteins.

Löwenadler, Björn; Nilsson, Björn; Abrahmsén, Lars; Moks, Tomas; Ljungqvist, L; Holmgren, Erik; Paléus, Sven; Josephson, Staffan; Philipson, Lennart; Uhlén, Mathias

doi:10.1002/j.1460-2075.1986.tb04509.x

Cited by 74 publications

(26 citation statements)

References 35 publications

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“…The 2.1-kb KpnI-BglII fragment of YRpSDB25 was subcloned into pRIT2 (Lowenadler et al 1986) to give pJ100, which contains a protein A-Sdb25 fusion gene having all but the first 13 amino acid residues of the Sdb25 polypeptide. The fusion protein was induced in E. coli, purified using an IgG--Sepharose column (Pharmacia), and injected into rabbits.…”

Section: Preparation Of Sdb25 Antiserummentioning

confidence: 99%

P40SDB25, a putative CDK inhibitor, has a role in the M/G1 transition in Saccharomyces cerevisiae.

Donovan¹,

Toyn²,

Johnson³

et al. 1994

Genes Dev.

138

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The Saccharomyces cerevisiae protein kinase Dbf2 carries out an essential function in late mitosis, and its kinase activity is cell-cycle regulated around anaphase/telophase. We have isolated SDB25, a high copy suppressor of temperature-sensitive dbf2 mutants, and genetic analysis suggests that the two proteins may function in parallel pathways in late mitosis. SDB25 encodes p40, a previously characterized substrate and potent inhibitor of Cdc28 kinase activity. Sdb25 is a phosphoprotein, and Sdb25 immunoprecipitates have a histone H1 kinase activity that is CDC28-dependent. Remarkably, Sdb25 transcript levels, protein levels, and associated kinase activity are precisely cell-cycle regulated, sharing a common peak in late mitosis. Moreover, Sdb25 protein levels and associated kinase activity are sharply up-regulated at the peak of Dbf2 kinase activity in cells released from a dbf2 ts block. The Sdb25 protein then disappears around Start in the next cell cycle. This indicates that SDB25 function is confined to M/GI, and morphological analysis of sdb25A cells supports this conclusion. Our data suggest that Sdb25 functions in a pathway in late mitosis leading to the down-regulation of Cdc28 kinase activity as cells traverse the M/G1 boundary.

show abstract

Section: Preparation Of Sdb25 Antiserummentioning

confidence: 99%

P40SDB25, a putative CDK inhibitor, has a role in the M/G1 transition in Saccharomyces cerevisiae.

Donovan¹,

Toyn²,

Johnson³

et al. 1994

Genes Dev.

138

View full text Add to dashboard Cite

show abstract

“…Construction of expression plasmids, cDNA fragments of the New Guinea C strain of DEN-2 (Gruenberg et al, 1988) were inserted into the unique BamHI site of the plasmid expression vector pRIT2T (Nilsson et al, 1985;Lowenadler et al, 1986) purchased from Pharmacia LKB Biotechnology (Fig. 1).…”

Section: Cells and Virusmentioning

confidence: 99%

Processing of the dengue virus type 2 proteins prM and C-prM

Murray

Aaskov

Wright

1993

Journal of General Virology

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A glycoprotein C-prM of 35000 M r was immunoprecipitated from lysates ofAedes albopictus cells infected with dengue virus type 2 (DEN-2) using antisera directed against the C protein or an amino-terminal fragment of the prM glycoprotein. C-prM was not detected in infected Vero cells. The prM glycoprotein synthesized in infected A. albopictus and Vero cells was cleaved to produce the membrane-associated virion protein (M) and the non-M fragment (pr) immediately preceding or occurring simultaneously with the release of viral particles from cells. The cleavage was less efficient in mosquito cells. The pr fragment was found only in the medium and was not rapidly degraded. To obtain prspecific and M-specific antisera for these studies, proteins containing fragments of DEN-2 prM fused with staphylococcal Protein A were synthesized in Escherichia coli using the expression vector pRIT2T. The fusion proteins were stable and were used to raise antisera in rabbits for immunoprecipitation of radiolabelled cell extracts and culture medium. This is the first report of the detection of a C-prM protein in flavivirus-infected cells and the identification of the pr component of prM.

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“…Rabbit anti-azurin serum was obtained by immunization of white rabbits according to L6wenadler et al [15], with minor modifications.…”

Section: Immunological Techniquesmentioning

confidence: 99%

Expression of the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli

et al. 1989

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The structural gene for the blue copper protein azurin from Pseudomonas aeruginosa has been subcloned in different expression plasmid vectors. The highest yield of expression was obtained when the gene with its native ribosome-binding site was placed downstream of the lac promoter in plasmid pUCI8. The protein is exported to the periplasmic space in Escherichia coli and the amount corresponds to 27% of the total protein content in the periplasmic space. The preprotein is cleaved correctly according to N-terminal sequencing of the purified protein. Azurin has been purified in large amounts and is spectroscopically indistinguishable from the protein purified from P. aeruginosa.

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Production of specific antibodies against protein A fusion proteins.

Cited by 74 publications

References 35 publications

P40SDB25, a putative CDK inhibitor, has a role in the M/G1 transition in Saccharomyces cerevisiae.

P40SDB25, a putative CDK inhibitor, has a role in the M/G1 transition in Saccharomyces cerevisiae.

Processing of the dengue virus type 2 proteins prM and C-prM

Expression of the blue copper protein azurin from Pseudomonas aeruginosa in Escherichia coli

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