Protein phosphorylation and dephosphorylation are the general means of regulation metabolism within a cell. A PKA catalytic subunit was found in Arabidopsis genome using Blast software. The cDNA was cloned by RT-PCR a...Protein phosphorylation and dephosphorylation are the general means of regulation metabolism within a cell. A PKA catalytic subunit was found in Arabidopsis genome using Blast software. The cDNA was cloned by RT-PCR and sequencing result indicated a high degree of homology at protein level. The cDNA was subcloned into pET30a (+) and expressed in E. coli at different temperatures. The target protein was insoluble when induced at 37degreesC, while dissolvable if induced at 22degreesC with 0.01 mmol/L IPTG. Ni2+-NTA affinity chromatography was used to purify the target protein, which was shown to have cAMP-dependent protein kinase activity. Western blotting analysis indicated that stress treatments affected the expression of PKA catalytic subunit at protein level just to a small extent.展开更多
文摘Protein phosphorylation and dephosphorylation are the general means of regulation metabolism within a cell. A PKA catalytic subunit was found in Arabidopsis genome using Blast software. The cDNA was cloned by RT-PCR and sequencing result indicated a high degree of homology at protein level. The cDNA was subcloned into pET30a (+) and expressed in E. coli at different temperatures. The target protein was insoluble when induced at 37degreesC, while dissolvable if induced at 22degreesC with 0.01 mmol/L IPTG. Ni2+-NTA affinity chromatography was used to purify the target protein, which was shown to have cAMP-dependent protein kinase activity. Western blotting analysis indicated that stress treatments affected the expression of PKA catalytic subunit at protein level just to a small extent.
