Expression and biology identification of the human epididymis-specific gene ESC42 in E. coli.
- Author:
Xiao-Fang SHEN
1
;
Jian-Yuan LI
;
Hai-Yan WANG
;
Jiang-Nan XUE
;
Qi-Zhi CAO
;
Pei-Wen LIAN
;
Hui CHEN
;
Mei-Na XIE
Author Information
- Publication Type:Journal Article
- MeSH: Amino Acid Sequence; Animals; Antibodies, Monoclonal; immunology; Cloning, Molecular; Defensins; biosynthesis; genetics; immunology; Epididymis; metabolism; Escherichia coli; genetics; Gene Library; Humans; Male; Mice; Mice, Inbred BALB C; Plasmids; genetics; Recombinant Fusion Proteins; biosynthesis; immunology
- From: National Journal of Andrology 2005;11(2):106-111
- CountryChina
- Language:Chinese
-
Abstract:
OBJECTIVETo provide materials for the study of the function of ESC42 protein specifically expressed in the human epididymis.
METHODSThe ESC42 gene was amplified from the human epididymis cDNA library by PCR and then cloned into prokaryotic expression vector pGEX-4T-1, expressed and purified by recombinant DNA techniques. The specificity of ESC42 protein was identified by Western blot and MALDI-TOF-MS. The database was searched by Ms-Fit.
RESULTSThe recombinant plasmid expressed a Mr 38 x 10(3) fusion protein in E. coli at a level of 30% of the total protein, and the purity was as high as 99%. The ESC42 protein was identified by ESC42 monoclonal antibody and its molecular weight was 11 978.12, tested by MALDI-TOF-MS. The peptide mass fingerprint analysis showed that the coverage rate of the sequence reached 48% with 100% matching. The motif scan in Prosite database reveal that ESC42 belonged to the beta-defensin family and had antibacterial activity.
CONCLUSIONObtaining high purity of rhESC42 protein may lay a foundation for the study of its functions.
