Immunoaffinity purification of glucose/xylose isomerase from Streptomyces.
A procedure was developed to purify glucose/xylose isomerase from cell extract of Streptomyces sp. NCIM 2730 using immunoaffinity chromatography. High-titer polyclonal antibodies were raised in rabbit using electrophoretically homogeneous glucose/xylose isomerase as an antigen. The specificity of antibodies was confirmed by double immunodiffusion, rocket electrophoresis, and Western-blot ELISA, which revealed the presence of a single immunoreactive protein with an Mr of 40,000. The antibodies recognized 2-3 antigenic determinants/mol of enzyme and were found to partially neutralize the enzymatic activity in an immunotitration experiment. The affinity gel was prepared by coupling antibodies at pH 10.0 to divinyl sulfone-activated Sepharose CL-4B. The glucose/xylose isomerase purified by immunoaffinity chromatography yielded 75% recovery with a single enzymatically active protein band on gel electrophoresis and showed specific activity of 16 U/mg. The crossreaction of the antibodies with glucose isomerase from other actinomycetes indicated that they share common epitopes.
Ghatge
M
M
Division of Biochemical Sciences, National Chemical Laboratory, Pune, India.
Mawal
Y
Y
Gaikwad
S
S
Deshpande
V
V
D019747:Aldose-Ketose Isomerases; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D002238:Carbohydrate Epimerases / Q000276:immunology / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods*; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D005779:Immunodiffusion; D011817:Rabbits; D013302:Streptomyces / Q000201:enzymology*
D000939:Epitopes; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase