Prokaryotic Expression And Purification Of The Arabidopsis Auxin Binding Protein1(ABP1)

Auxin is an essential phytohormone in the process of plant growth and development. Auxin regulates the plant embryogenesis, morphogenesis, organ formation and other reproductive processes. It regulates the cell division, growth, differentiation and transduction of signals in cellular level. As a binding protein to auxin, auxin-binding protein1(ABP1) is paid much attention. However, more researches are needed to make it clear that how ABP1takes part in the auxin signal transduction by collaborating with other proteins. The ABP1protein has a specific binding on auxin. In this research, the ABP1of high purity was obtained by prokaryotic expression, which laid a foundation for the following study of ABP1:the molecular structure, auxin signal transduction mechanism, and the construction of biosensors using ABP1as the recognizing molecule.The research studied the prokaryotic expression of Arabidopsis ABP1and the protein purification. The results are as follows:1. Primers were designed based on ABP1cDNA sequence published in TAIR database. The ABP1gene was cloned by RT-PCR, the sequencing result was consistent with that in TAIR database.2. The ABP1gene was cloned into pGEX4T-1(Prokaryotic expression vector) successfully, the positive recombinant was selected by double digestion.3. The recombinant pGEX4T-1-ABP1was transformed into competent Escherichia coli BL21(DE3) and then induced by isopropy1p-D-1-thiogalactopyranoside(IPTG) under different inducing time, temperature, IPTG density. The optimized inducing condition for fusion protein expression was:Escherichia coli BL21(DE3) was induced for10hours under25℃with an IPTG concentration at0.75mmol/L4. The protein was obtained and purified with GST affinity column using one-step protocol, the concentration of the target protein was also determined.