肌钙蛋白Ⅰ的可溶性表达及纯化研究-东南大学学报医学版

肌钙蛋白Ⅰ的可溶性表达及纯化研究

单位：南京大学医药生物技术国家重点实验室, 江苏南京 210093

分类号：Q786; R322.74

出版年·卷·期（页码）：2009·28·第一期（1-3）

摘要：

目的: 提高肌钙蛋白Ⅰ(troponin Ⅰ,Tn Ⅰ)在大肠杆菌中的可溶性表达并进行分离纯化。方法和结果: 将质粒CBD-intein/pTWIN1和Tn Ⅰ/pET28a分别用Nco Ⅰ及Xho Ⅰ双酶切,回收。回收后的载体CBD-intein/pTWIN1与Tn Ⅰ片段用T4 连接酶连接,得到重组质粒CBD-intein-Tn Ⅰ/pTWIN1。将重组质粒转化大肠杆菌BL21(DE3),经IPTG诱导表达得到融合蛋白CBD-intein-Tn Ⅰ。经chitin亲和层析和intein介导的融合蛋白自我裂解,纯化得到重组Tn Ⅰ。结论: 通过CBD-intein融合表达系统一步式裂解分离纯化能方便快捷地获得可溶性的Tn Ⅰ。

Objective To highly express soluble recombinant troponin Ⅰ (Tn Ⅰ) in E.coli and obtain purified protein-Tn Ⅰ.Method and Result The Tn Ⅰ gene was obtained by digesting the plasmid Tn Ⅰ-pET28a using restriction enzymes Nco Ⅰ and Xho Ⅰ,the purified Tn Ⅰ was inserted into Nco Ⅰ / Xho Ⅰ sites of CBD-intein/pTWIN1 to form a recombinant expression plasmid CBD-intein-Tn Ⅰ/pTWIN1,then transformed it into E.coli for expression.The fusion protein CBD-intein-Tn Ⅰ was expressed by addition of IPTG and purified by chitin affinity chromatography.Conclusion The soluble Tn Ⅰ can conveniently obtain by CBD-intein expression system and chitin chromatography.

参考文献：

[1] EBASHI S,KODAMA A,EBASHI F.Troponin Ⅰ preparation and physiological function[J].Biochem,1968,64:465-477.
[2] MOSES M A,WIEDERSCHAIN D,WU I,et al.Troponin Ⅰ is present in human cartilage and inhibits angiogenesis[J].Proc Natl Acad Sci USA,1999,96:2645-2650.
[3] WADMAN M.Anticancer activity of endostatin redeemed[J].Nature,1999,397(6720):549.
[4] FELDMAN L,ROULEAU C.Troponin Ⅰ inhibits capillary endothelial cell proliferation by interaction with the cell's bFGF receptor[J].Microvascular Research,2002,63:41-49.
[5] KERN B E,BALCOM J H,ANTONIU B A,et al.Troponin I peptide (Glu 94-Leu 123),a cartilage-derived angiogenesis inhibitor:in vitro and in vivo effects on human endothelial cells and on pancreatic cancer[J].Gastrointest Surg,2003,7(8):961-969.
[6] 颜明,华子春.人快速收缩骨骼肌肌钙蛋白Ⅰ在大肠杆菌中的表达及建立其快速复性方法[J].东南大学学报:医学版,2005,24(1):36-38.

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