[Efficient fusion expression of G13 domain derived from granulysin in Escherichia coli].
The G13 domain derived from granulysin shows high antimicrobial activities against Gram-positive and Gram-negative bacteria but does not lyse Jurkat cells or liposomes. To explore a new approach for high expression of the G13 domain, we fused the sequence encoding G13 to thioredoxin (Trx) gene to construct the recombinant expression vector (pThioHisA-G13). A cyanogen bromide (CNBr) cleavage site was introduced between the Trx and G13 to facilitate final release of the recombinant G13. The recombinant expression vector, pThioHisA-G13, was transformed into E. coli BL21 (DE3). Upon induction by IPTG Trx-G13 fusion protein was expressed and took the form of inclusion bodies counting 58% (W/W) of total cellular proteins. The inclusion body was solved by urea (8 mol/L) and then cleaved by CNBr. We purified the recombinant peptide G13 by one-step cation exchange chromatography. Results of agarose diffuse assay analysis indicated that the recombinant G13 exhibited antibacterial activity. The procedure described in this study will provide a reliable and simple method for highly efficient production of some cationic antimicrobial peptides.
Liu
Xiaoqiang
X
Key Laboratory of Ecological Engineering and Biotechnology of Anhui Province, School of Life Science, Anhui University, Hefei 230039, China.
Zha
Xiangdong
X
Xiao
Yazhong
Y
Yang
Jinhuan
J
Li
Nengshu
N
D000890:Anti-Infective Agents / Q000378:metabolism; D000945:Antigens, Differentiation, T-Lymphocyte / Q000235:genetics*; D003488:Cyanogen Bromide / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000096:biosynthesis* / Q000235:genetics; D002479:Inclusion Bodies / Q000378:metabolism; D017434:Protein Structure, Tertiary / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics; D013879:Thioredoxins / Q000235:genetics; D014162:Transfection
D000890:Anti-Infective Agents; D000945:Antigens, Differentiation, T-Lymphocyte; C075005:GNLY protein, human; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D044365:GTP-Binding Protein alpha Subunits, G12-G13; D003488:Cyanogen Bromide