Prokaryotic expression and renaturation of engineering chimeric Fab antibody against human hepatoma.
OBJECTIVE
To express chimeric Fd (cFd) and chimeric light chain (cL) in E.coli respectively and refold them into chimeric Fab (cFab) antibody.


METHODS
cFd and cL genes were respectively inserted into the prokaryotic expression vector pET32a to construct recombinant vectors pET32a/cFd and pET32a/cL. Then, the competent E.coli cells were transformed by the recombinant vectors and induced by IPTG. Moreover, a large quantity of cFd and cL expression products were prepared and mixed with equal molar to refold into cFab by gradient dialysis. The refolded products were identified and analyzed by sodium SDS-PAGE, Western blotting, ELISA and HPLC.


RESULTS
High efficient prokaryotic expressions of both cFd and cL in the form of non-fusion protein were obtained with the expression levels of 28.3% and 32.3% of total bacteria proteins, respectively. Their relative molecular masses were all 24 ku or so, and both of them mainly existed in the form of inclusion bodies. In addition, cFd and cL were successfully refolded into cFab by gradient dialysis, with about 59.45% of recovery when the starting total protein concentration was 100 microg/mL. The renatured cFab could specifically bind to related antigen with high affinity.


CONCLUSIONS
The cFab antibody against human hepatoma was highly and efficiently expressed and refolded, which laid a solid foundation for studying its application in the treatment of hepatoma.
Xing
Jin-Liang
JL
Cell Engineering Research Center, Faculty of Preclinical Medicine, Fourth Military Medical University, Xi'an 710032, Shaanxi Province, China.
Yang
Xiang-Min
XM
Yao
Xi-Ying
XY
Song
Fei
F
Chen
Zhi-Nan
ZN
D001698:Biomedical Engineering; D006528:Carcinoma, Hepatocellular / Q000235:genetics* / Q000276:immunology; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics* / Q000276:immunology; D008113:Liver Neoplasms / Q000235:genetics* / Q000276:immunology; D009695:Nucleic Acid Renaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000276:immunology
D007140:Immunoglobulin Fab Fragments; D011993:Recombinant Fusion Proteins