Hirudin as a novel fusion tag for efficient production of lunasin in Escherichia coli.
Fusion expression provides an effective means for the biosynthesis of longer peptides in Escherichia coli. However, the commonly used fusion tags are primarily suitable for laboratory scale applications due to the high cost of commercial affinity resins. Herein, a novel approach exploiting hirudin as a multipurpose fusion tag in combination with tobacco etch virus (TEV) protease cleavage has been developed for the efficient and cost-effective production of a 43-amino acid model peptide lunasin in E. coli at preparative scale. A fusion gene which allows for lunasin to be N-terminally fused to the C-terminus of hirudin through a flexible linker comprising a TEV protease cleavage site was designed and cloned in a secretion vector pTASH. By cultivation in a 7-L bioreactor, the fusion protein was excreted into the culture medium at a high yield of ~380 mg/L, which was conveniently recovered and purified by inexpensive HP20 hydrophobic chromatography at a recovery rate of ~80%. After polishing and cleavage with TEV protease, the finally purified lunasin was obtained with ≥95% purity and yield of ~86 mg/L culture medium. Conclusively, this hirudin tagging strategy is powerful in the production of lunasin and could be applicable for the production of other peptides at preparative scale.
a Department of Molecular Biology, State Key Laboratory of Natural Medicines, School of Life Science and Technology , China Pharmaceutical University , Nanjing , P.R. China.
Qinghua
Q
Tian
a Department of Molecular Biology, State Key Laboratory of Natural Medicines, School of Life Science and Technology , China Pharmaceutical University , Nanjing , P.R. China.
Ping
P
Zhang
a Department of Molecular Biology, State Key Laboratory of Natural Medicines, School of Life Science and Technology , China Pharmaceutical University , Nanjing , P.R. China.
Zhan
Z
Gao
a Department of Molecular Biology, State Key Laboratory of Natural Medicines, School of Life Science and Technology , China Pharmaceutical University , Nanjing , P.R. China.
Hengli
H
Li
a Department of Molecular Biology, State Key Laboratory of Natural Medicines, School of Life Science and Technology , China Pharmaceutical University , Nanjing , P.R. China.
Zhengli
Z
Bai
a Department of Molecular Biology, State Key Laboratory of Natural Medicines, School of Life Science and Technology , China Pharmaceutical University , Nanjing , P.R. China.
Shuhua
S
Tan
D045744:Cell Line, Tumor; D003001:Cloning, Molecular / Q000379:methods*; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D006629:Hirudins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism; D030262:Soybean Proteins / Q000235:genetics* / Q000378:metabolism; D013025:Glycine max / Q000235:genetics* / Q000378:metabolism
C120514:GM2S-1 protein, soybean; D006629:Hirudins; D011993:Recombinant Fusion Proteins; D030262:Soybean Proteins; D010450:Endopeptidases; C099488:TEV protease
Escherichia coli; fusion expression; fusion tag; hirudin; hydrophobic chromatography; lunasin