Versatile E. coli thioredoxin specific monoclonal antibodies afford convenient analysis and purification of prokaryote expressed soluble fusion protein

ABSTRACT A large portion of the human pro-opiomelanocortin (POMC) peptide corresponding to amino acid residues 59–241 has been cloned and expressed in Escherichia coli. A 1·0 kb DNA fragment encoding this peptide was cloned into the expression vectors pUC8 and pUR291. Plasmid pJMBG51 (a pUC8 recombinant) was found to direct the expression of a 24 kDa peptide. The recombinant pUR291 (pJMBG52) was shown to produce a β-galactosidase fusion protein of 140 kDa. Western blot analysis showed that both the 24 kDa and 140 kDa peptides are recognized by antibodies raised against POMC-derived peptides. The β-galactosidase fusion protein has been partially purified from crude E. coli cell lysates using affinity chromatography on p-aminobenzyl-1-thio-β-d-galactopyranoside agarose.

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Monoclonal antibodies specific to a Ca2+-bound form of lipocortin I distinguish its Ca2+-dependent phospholipid-binding ability from its ability to inhibit phospholipase A2

Biochemical Journal ◽

10.1042/bj2690709 ◽

1990 ◽

Vol 269 (3) ◽

pp. 709-715 ◽

Cited By ~ 11

Author(s):

H Hayashi ◽

M K Owada ◽

S Sonobe ◽

K Domae ◽

T Yamanouchi ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Phospholipase A2 ◽

Inhibitory Activity ◽

Biological Effects ◽

Free Form ◽

E Coli ◽

Phospholipid Binding ◽

Ef Hand

Lipocortin I, a Ca2(+)-and phospholipid-binding protein without EF-hand structures, has many biological effects in vitro. Its actual role in vivo, however is unknown. We obtained and characterized five monoclonal antibodies to lipocortin I. Two of these monoclonal antibodies (L2 and L4-MAbs) reacted with the Ca(+)-bound form of lipocortin I, but not with the Ca2(+)-free form, both in vivo and in vitro. Lipocortin I required greater than or equal to 10 microM-Ca2+ to bind the two antibodies, and this Ca2+ requirement was not affected by phosphatidylserine. L2-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I and inhibited its binding to Escherichia coli membranes and to phosphatidylserine in vitro. L4-MAb abolished the phospholipase A2 inhibitory activity of lipocortin I, but did not affect its binding to E. coli membranes or to phosphatidylserine. These findings indicated that the inhibition of phospholipase A2 by lipocortin I was not simply due to removal or capping of the substrates in E. coli membranes. Furthermore, an immunofluorescence study using L2-MAb showed the actual existence of Ca2(+)-bound form of lipocortin I in vivo.

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Generation of Monoclonal Antibodies to the AML1-ETO Fusion Protein: Strategies for Overcoming High Homology

Hybridoma ◽

10.1089/hyb.2011.0037 ◽

2011 ◽

Vol 30 (5) ◽

pp. 433-443 ◽

Cited By ~ 1

Author(s):

Stephen G. Simkins ◽

Steven L. Knapp ◽

George H. Brough ◽

Karen L. Lenz ◽

Lise Barley-Maloney ◽

...

Keyword(s):

Monoclonal Antibodies ◽

Fusion Protein ◽

High Homology

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Probing the Flavivirus Membrane Fusion Mechanism by Using Monoclonal Antibodies

Journal of Virology ◽

10.1128/jvi.01041-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 11526-11531 ◽

Cited By ~ 34

Author(s):

Karin Stiasny ◽

Samantha Brandler ◽

Christian Kössl ◽

Franz X. Heinz

Keyword(s):

Monoclonal Antibodies ◽

Fusion Protein ◽

Binding Sites ◽

Fusion Inhibition ◽

Tick Borne Encephalitis ◽

Target Membrane ◽

Initial Interaction ◽

Tick Borne Encephalitis Virus ◽

Late Stages

ABSTRACT In this study, we investigated in a flavivirus model (tick-borne encephalitis virus) the mechanisms of fusion inhibition by monoclonal antibodies directed to the different domains of the fusion protein (E) and to different sites within each of the domains by using in vitro fusion assays. Our data indicate that, depending on the location of their binding sites, the monoclonal antibodies impaired early or late stages of the fusion process, by blocking the initial interaction with the target membrane or by interfering with the proper formation of the postfusion structure of E, respectively. These data provide new insights into the mechanisms of flavivirus fusion inhibition by antibodies and their possible contribution to virus neutralization.

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Expression Analysis and Nuclear Import Study of Full-length Isoforms Importin α as 6x Histidin-tagged Fusion Protein on the Intracellular Localization of Recombinant HBV Core Protein

Indonesian Journal of Biotechnology ◽

10.22146/ijbiotech.7412 ◽

2015 ◽

Vol 10 (1) ◽

Author(s):

Aris Haryanto

Keyword(s):

Fusion Protein ◽

Nuclear Import ◽

Recombinant Dna ◽

Core Protein ◽

Intracellular Localization ◽

Full Length ◽

Nuclear Pore ◽

E Coli ◽

Importin Α ◽

Localization Signal

Isoform importin α molecules play a central role in the classical nuclear import pathway, that occurs throughthe nuclear pore complex (NPC) and typically requires a specific nuclear localization signal (NLS). In this study,it was investigated the role of isoforms importin α in the nuclear import of wild type recombinant hepatitis B viruscore protein (WT rHBc), phosphorylated recombinant HBV core (rHBc) and recombinant HBV core without NLSby co-immunoprecipitation. Four recombinant full-length isoforms importin α as 6x histidin-tagged fusion proteinwere expressed and analysed from expression plasmid vectors Rch1, pHM 1969, pHM 1967 and pHM 1965. Theresults indicated that importin α-1, importin α-3, importin α-4 and importin α-5 can be expressed and isolatedfrom E. coli transformed recombinant DNA plasmid as protein in size around 58-60 kDa. By the nuclear transportstudy shown that isoforms importin α are involved in the nuclear import of WT rHBc, phosphorylated rHBc andrHBc without NLS. It also indicated that they have an important role for nuclear transport of from cytoplasm intothe nucleus.Keywords: NPC, NLS, importin α, importin β, isoforms importin α as 6x histidin-tagged fusion protein, WTrHBc, SV40 Tag, co-immunoprecipitation, westernblotting.

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