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Topic

Affinity chromatography

About: Affinity chromatography is a(n) research topic. Over the lifetime, 21907 publication(s) have been published within this topic receiving 667388 citation(s). The topic is also known as: Chromatography, Affinity.


Papers
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Journal ArticleDOI
15 Jul 1988-Gene
TL;DR: Plasmid expression vectors have been constructed that direct the synthesis of foreign polypeptides in Escherichia coli as fusions with the C terminus of Sj26, a 26-kDa glutathione S-transferase (GST; EC 2.5.1.18) encoded by the parasitic helminth Schistosoma japonicum.
Abstract: Plasmid expression vectors have been constructed that direct the synthesis of foreign polypeptides in Escherichia coli as fusions with the C terminus of Sj26, a 26-kDa glutathione S-transferase (GST; EC 2.5.1.18) encoded by the parasitic helminth Schistosoma japonicum. In the majority of cases, fusion proteins are soluble in aqueous solutions and can be purified from crude bacterial lysates under non-denaturing conditions by affinity chromatography on immobilised glutathione. Using batch wash procedures several fusion proteins can be purified in parallel in under 2 h with yields of up to 15 micrograms protein/ml of culture. The vectors have been engineered so that the GST carrier can be cleaved from fusion proteins by digestion with site-specific proteases such as thrombin or blood coagulation factor Xa, following which, the carrier and any uncleaved fusion protein can be removed by absorption on glutathione-agarose. This system has been used successfully for the expression and purification of more than 30 different eukaryotic polypeptides.

5,960 citations

Journal ArticleDOI
TL;DR: It is shown that nitric oxide synthetase activity requires calmodulin, and the native enzyme appears to be a monomer.
Abstract: Nitric oxide mediates vascular relaxing effects of endothelial cells, cytotoxic actions of macrophages and neutrophils, and influences of excitatory amino acids on cerebellar cyclic GMP. Its enzymatic formation from arginine by a soluble enzyme associated with stoichiometric production of citrulline requires NADPH and Ca2+. We show that nitric oxide synthetase activity requires calmodulin. Utilizing a 2',5'-ADP affinity column eluted with NADPH, we have purified nitric oxide synthetase 6000-fold to homogeneity from rat cerebellum. The purified enzyme migrates as a single 150-kDa band on SDS/PAGE, and the native enzyme appears to be a monomer.

3,175 citations

Journal ArticleDOI
06 Dec 1990-Nature
TL;DR: It is shown that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen and that rare phage can be isolated after affinity chromatography.
Abstract: NEW ways of making antibodies have recently been demonstrated using gene technology. Immunoglobulm variable (V) genes are amplified from hybridomas or B cells using the polymerase chain reaction, and cloned into expression vectors. Soluble antibody fragments secreted from bacteria are then screened for binding activities (see ref. 1 for review). Screening of V genes would, however, be revolutionized if they could be expressed on the surface of bacteriophage. Phage carrying V genes that encode binding activities could then be selected directly with antigen. Here we show that complete antibody V domains can be displayed on the surface of fd bacteriophage, that the phage bind specifically to antigen and that rare phage (one in a million) can be isolated after affinity chromatography.

2,967 citations

Journal ArticleDOI
TL;DR: It is demonstrated that successful application of affinity chromatography in many cases will critically depend on placing the ligand at a considerable distance from the matrix backbone.
Abstract: The preparation of a number of agarose and polyacrylamide bead derivatives useful in the purification of proteins by affinity chromatography is described. These techniques permit (a) the attachment of ligands to the gel through extended hydrocarbon chains which place the ligand at varying distances from the gel matrix backbone; (b) the covalent attachment of ligands to agarose or polyacrylamide gels through amino, carboxyl, phenolic, or imidazole groups of the ligand; and (c) the preparation of adsorbents containing ligands attached by bonds which are susceptible to specific chemical cleavage, thus providing means of removing the intact protein-ligand complex from the affinity adsorbent. It is demonstrated that successful application of affinity chromatography in many cases will critically depend on placing the ligand at a considerable distance from the matrix backbone. Techniques are also described which provide important approaches and considerations in the insolubilization of peptides and proteins to agarose and polyacrylamide.

2,594 citations

Journal ArticleDOI
04 Dec 1970-Science
TL;DR: Plasminogen was prepared from human plasma by affinity chromatography on L-lysine-substituted Sepharose with a specific activity of 100 caseinolytic units per milligram of nitrogen.
Abstract: Plasminogen was prepared from human plasma by affinity chromatography on L-lysine-substituted Sepharose. Thirty milligrams of plasminogen, with a specific activity of 100 caseinolytic units (Committee on Thrombolytic Agents) per milligram of nitrogen, were obtained from 340 milliliters of plasma. This corresponds to over 200-fold purification from plasma. Disc-gel electrophoresis at pH 8.3 indicated seven distinct bands, all of which contained activity.

2,043 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202211
2021161
2020242
2019235
2018255
2017314