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Att B-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。
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 Att B sites were designed and used to create the Att B-TEV-FLAG-AIK fusion gene by overlapping PCR. The resulting fusion gene was cloned into the donor vector pDONR223 by att B and att P mediated recombination（BP reaction）, then, transferred into the destination vector pDEST15 by att L and att R 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 r TEV 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.
出处
《生物工程学报》
CAS
CSCD
北大核心
2015年第12期1753-1763,共11页
Chinese Journal of Biotechnology
基金
国家自然科学基金(No.81272582)资助~~
关键词
阳离子多肽
位点特异性重组
GST融合蛋白
诱导表达
亲和层析
抑瘤活性
cationic peptide
site-specific recombination
GST fusion protein
inducible expression
affinity purification
antitumor activity
