[Cloning, expression and identification of functional fragment rC3B of human complement C3 in E. Coli].
This study was purposed to verify the binding part of human complement C3 to complement receptor III (CRIII) in monocytes, the peptide rC3B, including the binding-site, was expressed, purified and identified. rC3B, the binding part of human complement C3 to CRIII, was selected by computer-aided modeling and summarizing researches published. Then, rC3B gene fragment was amplified by PCR, and cloned into prokaryotic vector pQE30a. The fusion protein rC3B was expressed in E.coli M15 and purified by Ni(2+)-chelating affinity chromatography. The activity of rC3B was identified by Western blot and adherence assay with monocytes. The results showed that rC3B fragment was obtained, and a prokaryotic expression vector pQE30-rC3B was constructed. rC3B was efficiently expressed and purified. In Western blot, the target protein showed the activity of binding with C3 antibody, while the purified protein showed the activity of adherence with monocytes. It is concluded that the recombinant C3B was obtained and identified, and this study lay the basis for the further functional analysis of C3.
Gan
Hui
H
Institute of Transfusion Medicine, Academy of Military Medical Sciences, Beijing 100850, China.
Zhou
Yong
Y
Sun
Ping
P
Zhu
Xiao-Xia
XX
Wang
Quan-Li
QL
Zhan
Lin-Sheng
LS
D003001:Cloning, Molecular; D003176:Complement C3 / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D006801:Humans; D016177:Macrophage-1 Antigen / Q000235:genetics* / Q000378:metabolism; D017463:Receptors, Complement 3b / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D003176:Complement C3; D016177:Macrophage-1 Antigen; D017463:Receptors, Complement 3b; D011994:Recombinant Proteins