重组人基质金属蛋白酶12的原核表达与纯化  

Prokaryotic Expression and Purification of Recombinant Human MMP-12

作　　者：孙卫国 张灵霞[1] 史阅韩 翟斐 孙雯娜 侯江厚 SUN Wei-Guo;ZHANG Ling-Xia;SHI Yue-Han;ZHAI Fei;SUN Wen-Na;HOU Jiang-Hou(Army Tuberculosis Prevention and Control Key Laboratory,Beijing Key Laboratory of New Techniques of Tuberculosis Diagnosis and Treatment,Institute for Tuberculosis Research,Eighth Medical Center of General Hospital of PLA,Beijing 100091;Kunming City Matermal and Child Health Hospital,Kunming 650013,China)

机构地区：[1]解放军总医院第八医学中心结核病研究所,全军结核病防治重点实验室,结核病诊疗新技术北京市重点实验室,北京100091 [2]昆明市妇幼保健院,云南昆明560013

出　　处：《生物技术通讯》2020年第1期56-60,共5页Letters in Biotechnology

基　　金：国家传染病防治重大项目(2013ZX-10003-006)。

摘　　要：目的:利用原核系统表达重组对人基质金属蛋白酶12(MMP-12)并纯化,获得高纯度的人MMP-12蛋白。方法:在MMP-12氨基酸序列的N端加入His标签和肠激酶位点序列,构建MMP-12融合蛋白的原核表达载体,通过表达和亲和纯化获得MMP-12融合蛋白,以肠激酶对融合蛋白进行酶切和二次纯化,获得高纯度的人MMP-12。结果:构建了pET-MMP-12表达载体,并在大肠杆菌中实现了稳定高效表达。重组融合蛋白MMP-12经亲和层析纯化后,相对分子质量为42×10^3,纯度约95%。利用肠激酶对融合蛋白MMP-12进行酶切,酶切效率接近100%,通过二次纯化获得了MMP-12,相对分子质量为40.8×10^3,纯度大于95%。结论:利用原核表达系统高效表达了MMP-12融合蛋白,通过亲和纯化和肠激酶酶切的方法可以获得高纯度的MMP-12,为后续抗体制备和配基筛选奠定了基础。Objective:To obtain high purify recombinant human matrix metalloproteinase-12(MMP-12)ex pressed by prokaryotic system.Methods:His tag and enterokinase site were added to the N-terminal of the ami no acid sequence of MMP-12 protein and the nuceleotide sequence was cloned into prokaryotic expression vector pET-24a.The constructed recombinant plasmid pET-MMP-12 was transformed to E.coli Rosetta(DE3)for expres sion by induction of IPTG.The MMP-12 protein was expressed and purified by metal chelate affinity chromatogra phy.After digested by enterokinase,the high-purity human MMP-12 protein was secondary purified with affinity chromatography.Results:pET-MMP-12 was constructed successfully and recombinant MMP-12 protein was ex pressed stably with a relative molecular mass of about 42×10^3 and purity of 95%.The fusion protein cleavage effi ciency was close to 100%with a molecular weight of 40.8×10^3 and purity of more than 95%.Conclusion:The MMP-12 protein was overexpressed in prokaryotic system and high purity MMP-12 protein can be obtained by second affinity purification after enterokinase digestion,laying a foundation for subsequent antibody preparation and li gand selection.

关 键 词：基质金属蛋白酶12 原核表达 肠激酶 纯化 

分 类 号：Q78[生物学—分子生物学]