Construction of ADAM28 gene prokaryotic expression vector and fusion expression in E.coli

AIM:To clone the tooth growth related gene ADAM28 by subtractive hybridization technique.METHODS:Our research is to construct high-level prokaryotic expression vector of ADAM28,induce its expression in E.coli,and purify ADAM28 protein.The protein coding region of ADAM28 was amplified by RT-PCR and cloned into pMD18-T vector to produce the new construction,pMD18-T-ADAM28.The cloned ADAM28 segment was cut out with two restriction enzymes and was cloned into the prokaryotic expression vector,pGEX-4T-1,to produce the expression vector pGEX-4T-ADAM28.The recombinant plasmid was transformed into E.coli DH5α.GST- ADAM28 fusion protein was obtained after induction of IPTG.The fusion protein was extracted and purified by SDS-PAGE.RESULTS:①The expression vector pGEX-4T-ADAM28 was constructed successfully.The coding region sequence of ADAM28 was correctly inserted into the expression vector by endonucleases digestion,PCR identification and DNA sequencing analysis. Its open reading frame was integra.Mutation was not detected.②GST-ADAM28 fusion protein was obtained by initial purification.A new protein band of 35.3 kDa appeared on SDS-PAGE after induction of IPTG.CONCLUSION:ADAM28 protein could be expressed in E.coli expression system and purified initially.which may be the foundation to further study of the functions of ADAM28 protein.