       Document 0813
 DOCN  M9440813
 TI    Large-scale immunoaffinity purification of recombinant soluble human
       antigen CD4 from Escherichia coli cells.
 DT    9404
 AU    Wells PA; Beiderman B; Garlick RL; Lyle SB; Martin JP Jr; Herberg JT;
       Meyer HF; Henderson SL; Eckenrode FM; Cancer and Infectious Diseases
       Research Services, Upjohn Company,; Kalamazoo, MI 49001.
 SO    Biotechnol Appl Biochem. 1993 Dec;18 ( Pt 3):341-57. Unique Identifier :
       AIDSLINE MED/94128252
 AB    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.
 DE    Antibodies, Monoclonal  Antigens, CD4/*ISOLATION & PURIF
       Chromatography, Affinity  Chromatography, Ion Exchange  Electrophoresis,
       Polyacrylamide Gel  Escherichia coli/*METABOLISM  Human  Recombinant
       Fusion Proteins/CHEMISTRY/ISOLATION & PURIF  Recombinant
       Proteins/ISOLATION & PURIF  Regression Analysis  JOURNAL ARTICLE

       SOURCE: National Library of Medicine.  NOTICE: This material may be
       protected by Copyright Law (Title 17, U.S.Code).

