Purification of secreted alpha-amylases by immunoaffinity chromatography with cross-reactive antibody.
Two isozymes of rice alpha-amylases expressed and secreted by recombinant yeast were purified by immunoaffinity chromatography by using cross-reactive antibody. Antibodies raised against partially purified barley alpha-amylase adsorbed rice alpha-amylases in fermentation broth by a cross-reaction. By use of these antibodies as ligands, rice alpha-amylases were concentrated and purified to a high degree in one-step immunoaffinity chromatography. Because of the differences in the contaminating impurities between the barley alpha-amylase (antigen) from barley malt and rice alpha-amylases (target protein) secreted from yeast, the high purity of eluted alpha-amylases was attained without the use of highly purified antigen for immunization. Utilization of cross-reactive antibodies in immunoaffinity chromatography is useful for the purification of recombinant proteins in the absence of a sufficient amount and high enough purity of the target proteins to be purified.
Katoh
S
S
Department of Synthetic Chemistry and Biological Chemistry, Kyoto University, Japan.
Terashima
M
M
D000906:Antibodies; D001709:Biotechnology; D002846:Chromatography, Affinity / Q000379:methods*; D003429:Cross Reactions; D005285:Fermentation; D017343:Genes, Plant; D001467:Hordeum / Q000201:enzymology; D007527:Isoenzymes / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D012275:Oryza / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000235:genetics; D000516:alpha-Amylases / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism
D000906:Antibodies; D007527:Isoenzymes; D011994:Recombinant Proteins; D000516:alpha-Amylases