Abstract:Abstract: The chitin elicitor receptor kinase 1 (CERK1) family of plant immune kinases are pattern recogni-tion receptors (PRRs) capable of sensing bacterial and fungal infections. By recognizing pathogen-associated molecular patterns (PAMPs), such as some pathogen polysaccharides, or symbiotic factors, these receptor pro-teins involve in induction of PAMP-triggered immunity (PTI) or plant-microbe symbiosis. To determine the three-dimensional structure of CERK1 kinase domain, the E. coli-pRSF prokaryotic expression systems of wild-type and mutant AtCERK1 proteins were constructed for overexpression of CERK1 from Arabidopsis. The AtCERK1 proteins with a 6× His tag were expressed and subsequently purified by nickel column affinity chromatography. Based on the biophysical and biochemical properties of AtCERK1, ion exchange chro-matography and gel filtration chromatography were performed for further purification of the AtCERK1 pro-teins. The resulting AtCERK1 proteins, up to 95% purity, were subjected to screening of crystallization con-ditions. The AtCERK1F460V mutant protein was finally crystallized in the solution containing 0.2 mol/L am-monium fluoride and 20% PEG3350. X-ray diffraction data of these crystals were collected at the highest resolution of 3.2 ?魡. In conclusion, the AtCERK1 protein was successfully obtained and crystallized. The preliminary crystallographic analysis of this protein will facilitate future investigation of the structural and functional basis of AtCERK1 mediated plant immunity.
引用本文:
牛成群, 杨芷茜, 林 洁, 明振华. 植物免疫激酶AtCERK1的重组表达与结晶研究[J]. 生命科学研究, 2022, 26(5): 386-395.
NIU Cheng-qun, YANG Zhi-qian, LIN Jie, MING Zhen-hua. Expression and Crystallization of Recombinant Plant Immune Kinase AtCERK1. Life Science Research, 2022, 26(5): 386-395.