Hirudin as a novel fusion tag for efficient production of lunasin inEscherichia coli

Abstract

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.

Lunasin functionally enhances LDL uptake via inhibiting PCSK9 and enhancing LDLR expression in vitro and in vivo

“…Lunasin was prepared by using recombinant DNA technology as previously described [ 48 ], and dissolved in saline. Minimum essential medium (MEM) and Opti-MEM as well as FBS were purchased from Gibco (Grand Island, NY, USA).…”

Lunasin attenuates oxidant‐induced endothelial injury and inhibits atherosclerotic plaque progression in ApoE−/−mice by up‐regulating heme oxygenase‐1viaPI3K/Akt/Nrf2/ARE pathway

“…Polypeptide lunasin was prepared by using recombinant technology as previously described (23). DMEM and Opti-MEM were purchased from Thermo Fisher Scientific (Waltham, MA, USA).…”

Lunasin Improves the LDL-C Lowering Efficacy of Simvastatin via Inhibiting PCSK9 Expression in Hepatocytes and ApoE−/− Mice

“…Simvastatin was obtained from Nanjing Duly Biotechnology Co., Ltd. (Nanjing, China). Lunasin was prepared in our laboratory by using recombinant DNA technology as previously described [39]. Dil-LDL was obtained from Yiyuan Biotechnologies (Guangzhou, China).…”