[Expression of human Fab antibody against keratin in E.coli and its renaturation].
OBJECTIVE
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.


CONCLUSIONS
The successfully prepared anti-keratin Fab with binding activity to human keratin laid a solid foundation for its further application.
Fan
Jian-yong
JY
Dermatology Center of Chinese PLA, Xijing Hospital, Fourth Military Medical University, Xi'an 710032, China. fanjianyongfmmu@sina.com
Wang
Gang
G
Li
Wei
W
Wu
Yuan-ming
YM
Liu
Yu-feng
YF
D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis* / Q000235:genetics; D002479:Inclusion Bodies / Q000378:metabolism; D007633:Keratins / Q000235:genetics / Q000276:immunology*; D020673:Protein Renaturation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D014170:Transformation, Genetic
D007140:Immunoglobulin Fab Fragments; D007128:Immunoglobulin Fragments; D007147:Immunoglobulin Light Chains; D011993:Recombinant Fusion Proteins; C100277:immunoglobulins, Fd; D007633:Keratins