重组阳离子抗肿瘤肽AIK的原核表达、纯化及活性测定

Expression, purification of recombinant cationic peptide AIK in Escherichia coli and its antitumor activity

摘要:

利用Gateway克隆技术构建重组抗瘤肽AIK的原核表达体系，建立表达及纯化重组AIK的最优条件，为深入研究和利用AIK奠定基础。首先，设计含AttB重组位点的引物，通过重叠PCR技术扩增出AttB-TEV-FLAG-AIK序列，利用BP重组反应将目的序列TEV-FLAG-AIK克隆到供体载体pDONR223中，构建入门载体，再通过LR重组反应，将目的序列转移到目的载体pDEST15中，构建GST-AIK融合蛋白原核表达质粒。随后，在BL21 (DE3) 工程菌中优化诱导融合蛋白表达的条件。以谷胱甘肽磁珠纯化GST-AIK融合蛋白，再以rTEV酶切除GST，获得FLAG-AIK重组蛋白。最后以MTS法检测FLAG-AIK对白血病细胞HL-60的细胞毒性。菌液PCR验证和测序分析表明成功构建了重组抗瘤肽AIK的入门质粒和原核表达质粒。在BL21 (DE3) 工程菌中实现了GST-AIK融合蛋白的高效可溶性表达。并测得在37 ℃下以0.1 mmol/L IPTG诱导工程菌 (OD600=1.0) 4 h，重组蛋白表达量占菌体总蛋白的30% 以上。经GST亲和层析、rTEV酶切除GST标签及二次GST亲和层析获得纯度高于95%的FLAG-AIK蛋白。MTS法测得所制备的FLAG-AIK蛋白抑瘤活性与化学合成的AIK相当。总之，本课题应用Gateway克隆系统成功构建了抗瘤肽AIK的原核表达质粒，实现了GST-AIK融合蛋白的高效可溶性表达，经亲和层析获得了有生物活性的重组AIK多肽，为后续深入研究和大规模制备奠定了基础。

Abstract:

AIK is a novel cationic peptide with potential antitumor activity. In order to construct the AIK expression vector by Gateway technology, and establish an optimal expression and purification method for recombinant AIK, a set of primers containing AttB sites were designed and used to create the AttB-TEV-FLAG-AIK fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by attB and attP mediated recombination (BP reaction), then, transferred into the destination vector pDEST15 by attL and attR mediated recombination (LR reaction). All the cloning was verified by both colony PCR and DNA sequencing. The BL21 E. coli transformed by the GST-AIK expression plasmid was used to express the GST-AIK fusion protein with IPTG induction and the induction conditions were optimized. GST-AIK fusion protein was purified by glutathione magnetic beads, followed by rTEV cleavage to remove GST tag and MTS assay to test the growth inhibition activity of the recombinant AIK on human leukemia HL-60 cells. We found that a high level of soluble expression of GST-AIK protein (more than 30% out of the total bacterial proteins) was achieved upon 0.1 mmol/L ITPG induction for 4 h at 37 °C in the transformed BL21 E. coli with starting OD600 at 1.0. Through GST affinity purification and rTEV cleavage, the purity of the resulting recombinant AIK was greater than 95%. And the MTS assays on HL-60 cells confirmed that the recombinant AIK retains an antitumor activity at a level similar to the chemically synthesized AIK. Taken together, we have established a method for expression and purification of recombinant AIK with a potent activity against tumor cells, which will be beneficial for the large-scale production and application of recombinant AIK in the future.