title: Large-scale immunoaffinity purification of recombinant soluble human antigen CD4 from Escherichia coli cells.
abstract: A large-scale immunoaffinity (IA) purification process was developed for the isolation of recombinant soluble antigen CD4 (sCD4) from Escherichia coli fermentations. The monoclonal antibody used for IA purification of sCD4 recognized a conformation-dependent epitope on the surface of domain 1 of CD4. IA chromatography was used to purify both sCD4-183, consisting of the N-terminal 183 amino acids of human CD4, and sCD4-PE40, a fusion protein consisting of the N-terminal 178 amino acids of CD4 and amino acids 1-3 and 253-613 of Pseudomonas exotoxin A (PE40). sCD4-183 was purified from E. coli cell pellets using cell disruption, protein solubilization, oxidation, Q-Sepharose anion-exchange and IA chromatography steps. sCD4-PE40 was purified from cell pellets using cell disruption, protein solubilization, oxidation, Cu(2+)-immobilized metal-affinity chromatography, anion-exchange and IA chromatography steps. The IA-purified sCD4 analogues demonstrated the correct apparent molecular masses on SDS/PAGE. The immobilized monoclonal antibody appeared to select for correctly folded CD4 protein, since sCD4-183 and sCD4-PE40 purified by the IA method bound human-immunodeficiency-virus glycoprotein gp120 (HIV gp120) in vitro. sCD4-PE40 purified by IA chromatography also inhibited protein synthesis in CV-1 cells expressing HIV gp120/160 at the cell surface. Relatively high recoveries of sCD4-183 and sCD4-PE40 were observed in the IA step of the purification process (71 and 79% recovery respectively). The results demonstrate that immobilized monoclonal antibodies directed against conformational epitopes may be used for rapid purification of gram amounts of correctly folded protein from mixtures of oxidized E. coli proteins.
authors.affiliation: Cancer and Infectious Diseases Research Services, Upjohn Company, Kalamazoo, MI 49001.
authors.forename: P A
authors.initials: PA
authors.lastname: Wells
authors.forename: B
authors.initials: B
authors.lastname: Beiderman
authors.forename: R L
authors.initials: RL
authors.lastname: Garlick
authors.forename: S B
authors.initials: SB
authors.lastname: Lyle
authors.forename: J P
authors.initials: JP
authors.lastname: Martin
authors.forename: J T
authors.initials: JT
authors.lastname: Herberg
authors.forename: H F
authors.initials: HF
authors.lastname: Meyer
authors.forename: S L
authors.initials: SL
authors.lastname: Henderson
authors.forename: F M
authors.initials: FM
authors.lastname: Eckenrode
mesh_terms: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism*; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000302:isolation & purification; D012044:Regression Analysis
chemical_list: D000911:Antibodies, Monoclonal; D015704:CD4 Antigens; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; C076613:recombinant soluble CD4