Cloning, Expression and Antibody Production of the Disintegrin Domain of Human Fertilin beta.
A cDNA for the disintegrin domain (hf279) was isolated by PCR from human testis cDNAs. DNA sequencing indicated that hf279 cDNA encoded 93 amino acid residues, and it was identical with the reported sequence of fertilin beta. An expression plasmid, pGEX hf279, was constructed by inserting hf279 cDNA into plasmid pGEX-4T-2 containing gst gene. The expression plasmid was introduced into E.coli BL21(DE3) cells and a substantial amount of soluble fused protein GST-HF93 was obtained by the expression strain HF93/BL21 induced with IPTG. SDS-PAGE analysis revealed that the GST-HF93 fusion protein had an apparent molecular weight of 38 kD and accumulated up to 50% of bacterial soluble proteins. The fusion protein was purified by glutathione S-transferase (GST) Sepharose 4B column (purity 90%) and digested by thrombin to obtain the purified HF93 peptide (purity 80%). Polyclonal antibodies were obtained from the serum of miceimmunized with purified HF93 which was isolated by GST Sepharose 4B column and SDS-PAGE. ELISA and Western blot analysis showed its specificity to HF93. Therefore this antibody can be used in further studies on the function of HF93.
Ding
Bei-Bei
BB
National Laboratory of Coutraceptives and Device Research, Shanghai Institute of Planned Parenthood Research, Shanghai 200032, China. shenwang@sippr.stc.sh.cn
Shen
Wei-Ying
WY
Jiang
Chu
C
Wang
Jian
J
Gao
Er-Sheng
ES
Shen
Qing-Xiang
QX