title: An efficient method for refolding the extracellular portion of CD147 from the total bacterial lysate.
abstract: 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 (CD147(EC)) is responsible for its functional interactions with different signaling molecules. Due to the existence of two disulfide bonds, CD147(EC) is mainly expressed as an inclusion body in Escherichia coli. Here, we report a convenient rapid-dilution refolding protocol that enables the refolding of CD147(EC) efficiently from total bacterial lysate instead of pure inclusion bodies. Using this method, over 25 mg of CD147(EC) can be purified from 1 l of bacterial culture in M9 medium. The refolded CD147(EC) 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 immunoglobulin domains of CD147(EC) individually. Interestingly, we noticed that little protein was produced for the C-terminal immunoglobulin (Ig) domain of CD147(EC) by bacteria in M9 medium, even though it was overexpressed in Luria-Bertani (LB) medium. However, when the pH of the bacterial culture in M9 medium was adjusted in accordance with that in LB medium during growth, comparable expression level could be achieved.
authors.affiliation: 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.
authors.forename: Fei
authors.initials: F
authors.lastname: Song
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.forename: Yifei
authors.initials: Y
authors.lastname: Li
authors.forename: Qiang
authors.initials: Q
authors.lastname: Ru
authors.forename: Xiaobai
authors.initials: X
authors.lastname: Ren
authors.forename: Bin
authors.initials: B
authors.lastname: Xia
authors.forename: Zhi-Nan
authors.initials: ZN
authors.lastname: Chen
mesh_terms: D051926:Basigin / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002457:Cell Extracts / Q000032:analysis / Q000737:chemistry*; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005109:Extracellular Matrix / Q000737:chemistry*; D006801:Humans; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D017510:Protein Folding; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D000095983:Bacterial Lysates
chemical_list: D051926:Basigin; D002457:Cell Extracts; D020780:Matrix Metalloproteinase 9; D011994:Recombinant Proteins; C030259:Broncho-Vaxom; D000095983:Bacterial Lysates