Expression, purification, and in vitro refolding of a humanized single-chain Fv antibody against human CTLA4 (CD152).
A human-derived single-chain Fv (scFv) antibody fragment specific against human CTLA4 (CD152) was produced at high level in Escherichia coli. The scFv gene was cloned from a phagemid to the expression vector pQE30 with a N-terminal 6His tag fused in-frame, and expressed as a 29 kDa protein in E. coli as inclusion bodies. The inclusion body of scFv was isolated from E. coli lysate, solubilized in 8M urea with 10mM dithiothreitol, and purified by ion-exchange chromatography. Method for in vitro refolding of the scFv was established. The effects of refolding buffer composition, protein concentration and temperature on the refolding yield were investigated. The protein was renatured finally by dialyzing against 3mM GSH, 1mM GSSG, 150 mM NaCl, 1M urea, and 50 mM Tris-Cl (pH 8.0) for 48 h at 4 degrees C, and then dialyzed against phosphate-buffered saline (pH 7.4) to remove remaining denaturant. This refolding protocol generated up to a 70% yield of soluble protein. Soluble scFv was characterized for its specific antigen-binding activity by indirect cellular ELISA. The refolded scFv was functionally active and was able to bind specifically to CTLA4 (CD152). The epitopes recognized by refolded anti-CTLA4 scFv do not coincide with those epitopes recognized by CD80/CD86.
Chen
Li-Hong
LH
Key Laboratory of Transplant Engineering and Transplant Immunology, Ministry of Health, West China Hospital, Sichuan University, Chengdu 610064, P.R. China.
Huang
Qiang
Q
Wan
Lin
L
Zeng
Ling-Yu
LY
Li
Sheng-Fu
SF
Li
You-Ping
YP
Lu
Xiao-Feng
XF
Cheng
Jing-Qiu
JQ
D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D000918:Antibody Specificity / Q000276:immunology; D015703:Antigens, CD; D000943:Antigens, Differentiation / Q000276:immunology; D060908:CTLA-4 Antigen; D015870:Gene Expression; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D002479:Inclusion Bodies / Q000737:chemistry / Q000276:immunology; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000943:Antigens, Differentiation; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; D007135:Immunoglobulin Variable Region; D011994:Recombinant Proteins