Cloning, expression, and identification of a novel class IIa bacteriocin in the Escherichia coli cell-free protein expression system.
The NB-C1 gene, acquired from the result of data mining of the lactic acid bacteria genome, is a novel potential class IIa bacteriocin gene with the characteristic YGNGVxC cluster. To produce soluble NB-C1 efficiently and overcome issues of protein toxicity, we adopted a GFP fusion strategy using an Escherichia coli cell-free protein expression system. We constructed the expression vector pIVEX2.4d-GFP-NB-C1, which was expressed in both the batch mode and the continuous exchange cell-free (CECF) systems. The amount of soluble fusion protein achieved from the CECF system (2.2 mg/ml) was approximately three times higher than that in the batch mode (0.73 mg/ml). The soluble fusion protein was purified via one-step Ni-NTA affinity chromatography, with a concentration of 0.26 mg/ml and a purity of 95%. The purified NB-C1 showed strong antimicrobial activity against the indicator bacteria Listeria monocytogenes.
State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, People's Republic of China. haiqinchen@jiangnan.edu.cn
Haiqin
H
Chen
Xie
X
Yan
Fengwei
F
Tian
Yuanda
Y
Song
Yong Q
YQ
Chen
Hao
H
Zhang
Wei
W
Chen
D000900:Anti-Bacterial Agents / Q000378:metabolism*; D001430:Bacteriocins / Q000235:genetics* / Q000378:metabolism*; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008089:Listeria monocytogenes / Q000187:drug effects*; D008826:Microbial Sensitivity Tests; D050296:Microbial Viability / Q000187:drug effects; D014176:Protein Biosynthesis; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000633:toxicity
D000900:Anti-Bacterial Agents; D001430:Bacteriocins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins