Cloning, expression in Escherichia coli, and purification of soluble recombinant duck interleukin-2.
Interleukin-2 (IL-2) is a vital cytokine secreted by activated T lymphocytes, and plays an important role in the regulation of cellular and immunity of animals. In this study, a gene encoding duck IL-2 was cloned and the soluble recombinant duck IL-2 (rDuIL-2) was expressed in Escherichia coli via fusion with glutathione S-transferase (GST). The results indicated that the GST-rDuIL-2 fusion protein expressed in E. coli Origami (DE3) was confirmed to be of about 40 kDa molecular mass by SDS-PAGE and western blotting. In order to produce soluble rDuIL-2 in a low-cost, nontoxic and high-level expression process, lactose was used as a substitute for Isopropyl-β-D-thiogalactopyranoside (IPTG) to induce the above recombinant strain Origami (pGEX-DuIL-2). By optimizing the expression conditions, the production of soluble GST-rDuIL-2 fusion protein was about 29% of total cellular soluble proteins, which was similar with IPTG used as inducer. The soluble GST-rDuIL-2 fusion protein was purified by one-step affinity chromatography, and GST was removed by thrombin. Then rDuIL-2 was purified by a second affinity chromatography. As a result, the 95% pure rduIL-2 was obtained, and the yield of rDuIL-2 was about 10.6 mg/l bacterial culture. The bioactivity of rduIL-2 was determined by lymphocyte proliferation assay in vitro. Our study provided a feasible and convenient approach to produce soluble and biologically active rDuIL-2, which would be used as an immunoadjuvants for enhancing vaccine efficacy.
School of Biotechnology Engineering, Jimei University, Xiamen, 361021, China. cuihongdu@jmu.edu.cn
Cuihong
C
du
Long
L
Han
Zhijun
Z
Xie
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004372:Ducks / Q000235:genetics / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification*; D007785:Lactose / Q000378:metabolism; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015533:Transcriptional Activation / Q000187:drug effects
D007376:Interleukin-2; D011993:Recombinant Fusion Proteins; D007785:Lactose