The construction of pET15b-EGFP and cnhanced green fluorescent protein expression and purification within BL21(DE_3)

Objective The expression of vector pET15b-EGFP was constructed and the expression and purification of EGFP was performed.Methods Using pLEGFP-C1 plasmid as template,full-length EGFP cDNA was amplified by polymerase chain reaction.Then the amplified EGFP cDNA was added single A at its 3' end by using the template-independent terminal transferase activity of Taq DNA polymerase when there was only dATP exsisted.The purified EGFP cDNA being added single Aat its 3' end was then ligated with pGEM-T easy vector,and the ligated products were transformed into DH5α.After the blue-white selection,a single white clone was picked to be propagated and identified,the correct recombinant plasmid was named after pGEM-T-EGFP.pGEM-T-EGFP and pET15b plasmids were further double-enzyme digested by XhoI and BamHI,respectively,the EGFP cDNA fragment and linearized pET15b fragment were purified by low melting point agarose electrophoresis and was ligated with Ligafast DNA ligation system.The resultant ligation product was transformed into DH5α.The positive clone was propagated and the recombinant plasmid was extracted for further identification and sequencing.The correct recombinat plasmid was named after pET15bEGFP.E.coli BL21(DE_(3)) was transformed with pET15b-EGFP,and induced with IPTG for protein expression.Fusion protein with an N-terminal His-tag could be purified by Ni~(2+)-resin affinity chromatography.Purified protein was identified by SDS-PAGE and Western blotting.Results It was confirmed by DNA sequencing that the EGFP cDNA sequence in recombinant plasmid pET15b-EGFP was in accordance with the EGFP cDNA sequence in plasmid pLEGFP-C1 which was purchased from Clontech company.The recombinant plasmid pET15b-EGFP were constructed successfully.EGFP was expressed efficiently after pET15b-EGFP had been transformed and induced in E.coli BL21(DE_(3)),the yield of the purified protein was approximately 66 mg/100ml bacteria medium.SDS-PAGE and Western blotting demonstrated that the purified protein was EGFP indead.Conclusions Construction of recombinat plasmid pET15b-EGFP and expression and purification of the EGFP provides a basis for the research on the penetrating capability of Cell-penetrating peptide-EGFP fusion protein.