Cloning, overexpression and purification of Bacillus subtilis elongation factor Tu in Escherichia coli.
To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His6 tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.
Kim
S I
SI
Hyonam Kidney Laboratory, Soon Chun Hyang University, Seoul, Korea. sung237@unitel.co.kr
Kim
H Y
HY
Kwak
J H
JH
Kwon
S H
SH
Lee
S Y
SY
D001412:Bacillus subtilis / Q000235:genetics*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D010444:Peptide Elongation Factor Tu / Q000235:genetics / Q000302:isolation & purification*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D006153:Guanosine Diphosphate; D005982:Glutathione Transferase; D010444:Peptide Elongation Factor Tu