[Expression, renaturation and activity of BAC5-scFv expressed as inclusion body in E.coli].
OBJECTIVE
To study the renaturation, purification and binding activity of scFv of anti-nasopharyngeal carcinoma monoclonal antibody(mAb) BAC(5) expressed as inclusion body in E.coli.


METHODS
The E.coli BL21(DE3) transformed with the pET 22b-scFv was cultured and pulvereged by ultrasonic cell disintegrator. The collected inclusion bodies were denatured with 8 mol/L urea and renatured by dilution refolding, step dialysis and gel filtration chromatography. Binding activity of renatured BAC(5)-scFv was determined by immunohistochemical staining and Western blot.


RESULTS
BAC(5)-scFv purified though Ni-NTA His Bind chromatographic clomn showed high purity. The highest proteins recovery rate was obtained through gel filtration chromatography. It was proved by Western blot and immunocytochemical staining that the renatured BAC(5)-scFv protein could specifically bind to CNE2 cells.


CONCLUSIONS
BAC(5)-scFv expressed as inclusion body retained good activity after being dissolved, purified and renatured, which paves the way for preparing large amount of BAC(5)-scFv to be used for the study of radioimmunoimaging and therapy of nasopharyngeal carcinoma.
Tang
Cai-hua
CH
Department of Nuclear Medicine, Zhongshan Medical College, Sun yat-sen University, Guangzhou 510080, China. tcaihua2004@126.com
Xiao
Xi-bin
XB
Wu
Tao
T
Liang
Chang-sheng
CS
Liu
Chang-zheng
CZ
D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007150:Immunohistochemistry; D002479:Inclusion Bodies / Q000378:metabolism*; D009303:Nasopharyngeal Neoplasms / Q000276:immunology; D017510:Protein Folding
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments