Expression, purification and characterization of soluble human interleukin-6 receptor alpha-chain and its mutant protein in Escherichia coli.
To investigate the function of the N-terminal immunoglobulin (Ig)-like domain of the human interleukin-6 receptor alpha-chain (hIL-6R), we constructed a soluble human interleukin-6 receptor (shIL-6R) (named EC05, amino acids 20-354) and soluble variants of the shIL-6R lacking the Ig-like domain (named EC70, amino acids 105-354). The two extracellular portions of hIL-6R were expressed as soluble fusion proteins with thioredoxin in Escherichia coli and purified by using Ni-NTA agarose. Western blot showed that purified proteins were immunoreactive with the antibody against hIL-6R. They also possessed specific binding activity with human interleukin-6 (hIL-6) in ELISA analysis.
Wang
Jing
J
Department of Molecular Immunology, Institute of Basic Medical Sciences, Beijing, 100850, P.R. China.
Yang
Zhenhui
Z
Shi
Ming
M
Li
Yan
Y
Guo
Ning
N
Shen
Beifen
B
Feng
Jiannan
J
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry*; D053698:Interleukin-6 Receptor alpha Subunit / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012995:Solubility
D007074:Immunoglobulin G; D053698:Interleukin-6 Receptor alpha Subunit; D011994:Recombinant Proteins