[Preparation and characterization of recombinant protein A affinity packing].
To obtain an excellent antibody purification medium, affinity chromatographic packing with recombinant staphylococcal protein A (rProtein A) was synthesized and verified. With E. coli cells harboring the recombinant plasmid, the rProtein A was expressed and purified, then was conjugated to epichlorohydrin-activated Sepharose 4 Fast Flow to prepare an affinity chromatographic packing. The performances of the packing were validated with rabbit antiurate oxidase. After the reaction, the concentration of rProtein A coupled to Sepharose 4 Fast Flow was 1.5 x 10(-4) mol/L. Scatchard analysis of the binding isotherm for IgG showed excellent binding capacity on the adsorbent, giving a dissociation constant (Kd) of 2.28 x 10(-7) mol/L and a theoretical maximum adsorption capacity of 20. 697 g/L. The identification showed the packing was stable in 0.1 mol/L NaOH solution at 1 h. By using the packing, the pure antibody exhibited on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was obtained from rabbit serum after one-step elution, with 96. 1% of yield and 19 mg IgG for one milliliter of gel. The research laid the foundation of the localization of rProtein A affinity packing.
College of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China.
Mingming
M
Guo
Junling
J
Wang
Yanzhuo
Y
Wu
Mingbo
M
Xu
Xiangdong
X
Gao
D000818:Animals; D002846:Chromatography, Affinity / Q000295:instrumentation*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics; D007074:Immunoglobulin G / Q000302:isolation & purification; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012685:Sepharose / Q000737:chemistry; D013205:Staphylococcal Protein A / Q000096:biosynthesis* / Q000235:genetics; D014503:Urate Oxidase / Q000276:immunology
D007074:Immunoglobulin G; D011994:Recombinant Proteins; D013205:Staphylococcal Protein A; C097574:sepharose fast flow SP; D012685:Sepharose; D014503:Urate Oxidase