| Auxin, the first phytohormone diseovered in plant, plays a critical role in many phases of plant growth and development. Up to now, researches on synthesis, transport and signaling have already achieved quite great progress. TIR1, Aux/IAAs and TIR1-Aux/IAA co-receptor show a highly specific affinity to auxin. This characeristic indicates the brighter prospect for application of them in auxin extraction and purification. For this goal, it is an important foundation to priduce a large amount of activated TIR1 and Aux/IAAs. In this paper, AtIAA7 and AtTIR1 were isolated from Arabidopsis through RT-PCR. Expression vectors pGEX-KG-IAA7, pGEX-KG-TIR1, pIEx-3-IAA7 and pIEx-3-TIR1 were constructed based on pGEX-KG and pIEx-3 containing GST tag. Then prokaryotic expression vectors were transformed into three E.coli strains named Rosetta, BL21 and Tuner. Expression and purification conditions of recombinant protein were developed. Results showed that the expression of GST-IAA7 was higher in Rosetta with 0.4 mmol/L IPTG at 25℃ and accounted for 1.6% of the total weight of the cells. Unfortunately, GST-TIR1 only existed in inclusion body although fusion protein was highly expressed in Rosetta with induced by 0.4 mmol/L IPTG at 25 ℃. GST-IAA7 was purified through GST SefiroseTM resin and PMSF can significantly enhance stability of GST-IAA7 recombination proteins in vitro. Finally, GST tag was digested from fusion protein by thrombin. After optimizing culture conditions of S2 Drosophila cells, eukaryotic expression vector pIEx-3-IAA7 was transformed into S2 Drosophila cells and stable cell lines was screened. Results showed that fetal bovine serum (FBS) has little effect on short term growth of S2 cells. Eukaryotic expression vector pIEx-IAA7 and screening plasmid pCoHygro were co-transfected into S2 cells, then stable cell lines were achieved after screened by hygromycin B. Finally, IAA7 recombinant protein was successfully isolated and purified from culture medium through GST SefiroseTM resin. |
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