An efficient method for refolding the extraceUular portion of CD147 from the total bacterial lysate  

作　　者：Fei Song Xin Zhang Yifei Li Qiang Ru Xiaobai Ren Bin Xia Zhi-Nan Chen 

机构地区：[1]Department of Cell Biology, Cell Engineering Research Centre, Cancer Biology of State Key Laboratory and State Key Discipline of Cell Biology, Fourth Military Medical University, Xi＇an 710032, China [2]Beijing Nuclear Magnetic Resonance Center, College of Chemistry and Molecular Engineering, and College of Life Science, Peking University, Beijing 100871, China

出　　处：《Acta Biochimica et Biophysica Sinica》2011年第11期900-908,共9页生物化学与生物物理学报（英文版）

基　　金：This work was supported by the grants from the National Natural Science Foundation of China （30900234 F. Song）, and the Major State Basic Research Development Program of China （973 Program; 2009CB521700 ZN Chen and B. Xia）.

摘　　要：CD147 is a widely expressed transmembrane protein that mediates signal transduction, and it plays important roles in many physiological and pathological processes, such as tumor invasion and metastasis. The extracellular portion of CD147 （CD147Ec） is responsible for its functional interactions with different signaling molecules. Due to the existence of two disulfide bonds, CD147Ec is mainly expressed as an inclusion body in Escherichia coli. Here, we report a convenient rapid-dilution refolding protocol that enables the refoiding of CD147EC efficiently from total bacterial lysate instead of pure inclusion bodies. Using this method, over 25 mg of CD147Ec can be purified from 1 1 of bacterial culture in M9 medium. The refolded CD147Ec is well folded as characterized by nuclear magnetic resonance （NMR）, and it can induce the expression of matrix metalloproteinase-9 in fibroblast cells. The described protocol is also applicable to the refolding of two immunoglobu-lin domains of CD147Ec individually. Interestingly, we noticed that little protein was produced for the C-terminal immunoglobulin （Ig） domain of CD147rc by bacteria in M9 medium, even though it was overexpressed in Luria-Bertani （LB） medium. However, when the pH of the bac-terial culture in M9 medium was adjusted in accordance with that in LB medium during growth, comparable expression level could be achieved.CD147 is a widely expressed transmembrane protein that mediates signal transduction, and it plays important roles in many physiological and pathological processes, such as tumor invasion and metastasis. The extracellular portion of CD147 （CD147Ec） is responsible for its functional interactions with different signaling molecules. Due to the existence of two disulfide bonds, CD147Ec is mainly expressed as an inclusion body in Escherichia coli. Here, we report a convenient rapid-dilution refolding protocol that enables the refoiding of CD147EC efficiently from total bacterial lysate instead of pure inclusion bodies. Using this method, over 25 mg of CD147Ec can be purified from 1 1 of bacterial culture in M9 medium. The refolded CD147Ec is well folded as characterized by nuclear magnetic resonance （NMR）, and it can induce the expression of matrix metalloproteinase-9 in fibroblast cells. The described protocol is also applicable to the refolding of two immunoglobu-lin domains of CD147Ec individually. Interestingly, we noticed that little protein was produced for the C-terminal immunoglobulin （Ig） domain of CD147rc by bacteria in M9 medium, even though it was overexpressed in Luria-Bertani （LB） medium. However, when the pH of the bac-terial culture in M9 medium was adjusted in accordance with that in LB medium during growth, comparable expression level could be achieved.

关 键 词：CD 147 extracellular domain protein refolding isotope labeling nuclear magnetic resonance 

分 类 号：Q786[生物学—分子生物学] TH776.2[机械工程—仪器科学与技术]