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Characterization of Glycoproteins with the Immunoglobulin Fold by X-Ray Crystallography and Biophysical Techniques
Published on: July 5, 2018
[Expression of human Fab antibody against keratin in E.coli and its renaturation]
Jian-yong Fan1, Gang Wang, Wei Li
1Dermatology Center of Chinese PLA, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. fanjianyongfmmu@sina.com
Aim:
To express Fd fragment and L chain of human anti-keratin Fab in E.coli BL21(DE3) respectively and obtain human anti-keratin Fab by renaturation in-vitro.
Methods:
Genes of L chain and Fd fragment of anti-keratin antibody from the plasmids p3MH/ Fab were subcloned into vector pET32a respectively. The recombinant plasmids pETL and pETFd were transformed into E.coli BL21(DE3) and induced to express with IPTG. Fd fragment and L chain inclusion bodies were solubilized and combined in equal molar ratio in the refolding solution. The renatured Fab was characterized by SDS-PAGE, Western blot and ELISA.
Results:
Fd fragment and L chain of human anti-keratin Fab were efficiently expressed. ELISA and Western blot showed that the renatured Fab could bind with human keratin.
Conclusion:
The successfully prepared anti-keratin Fab with binding activity to human keratin laid a solid foundation for its further application.
