Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies.
Specific antibodies are a reliable tool to examine protein expression patterns and to determine the protein localizations within cells. Generally, recombinant proteins are used as antigens for specific antibody production. However, recombinant proteins from mammals and plants are often overexpressed as insoluble inclusion bodies in Escherichia coli. Solubilization of these inclusion bodies is desirable because soluble antigens are more suitable for injection into animals to be immunized. Furthermore, highly purified proteins are also required for specific antibody production. Plastidic acetyl-CoA carboxylase (ACCase: EC 6.4.1.2) from Arabidopsis thaliana, which catalyzes the formation of malonyl-CoA from acetyl-CoA in chloroplasts, formed inclusion bodies when the recombinant protein was overexpressed in E. coli. To obtain the purified protein to use as an antigen, we applied preparative disk gel electrophoresis for protein purification from inclusion bodies. This method is suitable for antigen preparation from inclusion bodies because the purified protein is recovered as a soluble fraction in electrode running buffer containing 0.1% sodium dodecyl sulfate that can be directly injected into immune animals, and it can be used for large-scale antigen preparation (several tens of milligrams).
Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan.
Yuki
Y
Okegawa
Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan.
Masanori
M
Koshino
Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan.
Teruya
T
Okushima
Department of Bioresource and Environmental Sciences, Faculty of Life Sciences, Kyoto Sangyo University, Kamigamo Motoyama, Kita-ku, Kyoto 603-8555, Japan. Electronic address: motohas@cc.kyoto-su.ac.jp.
Ken
K
Motohashi
D000103:Acetyl-CoA Carboxylase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D052179:Antigens, Plant / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D002479:Inclusion Bodies / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D052179:Antigens, Plant; D029681:Arabidopsis Proteins; D011994:Recombinant Proteins; D000103:Acetyl-CoA Carboxylase
Antigen preparation; Electroosmotic flow; Inclusion bodies; Preparative disk gel electrophoresis; Redox-regulation; Thioredoxin