Application of preparative disk gel electrophoresis for antigen purification from inclusion bodies

• Published in: Protein expression and purification 2016-02, Vol.118, p.77-82
• Main Authors: Okegawa, Yuki, Koshino, Masanori, Okushima, Teruya, Motohashi, Ken
• Format: Article
• Language: English
• Subjects: Acetyl-CoA Carboxylase - genetics; Acetyl-CoA Carboxylase - isolation & purification; Acetyl-CoA Carboxylase - metabolism; Antigen preparation; Antigens, Plant - genetics; Antigens, Plant - isolation & purification; Antigens, Plant - metabolism; Arabidopsis - enzymology; Arabidopsis - genetics; Arabidopsis Proteins - genetics; Arabidopsis Proteins - isolation & purification; Arabidopsis Proteins - metabolism; Electroosmotic flow; Electrophoresis, Polyacrylamide Gel - methods; Escherichia coli - chemistry; Escherichia coli - genetics; Escherichia coli - metabolism; Gene Expression; Inclusion bodies; Inclusion Bodies - chemistry; Inclusion Bodies - genetics; Inclusion Bodies - metabolism; Preparative disk gel electrophoresis; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Redox-regulation; Thioredoxin
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2015.10.008

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). •AccD protein was overexpressed as inclusion bodies in Escherichia coli.•AccD was purified from inclusion bodies by preparative disk gel electrophoresis.•Preparative disk gel electrophoresis is a useful method for antigen preparation.