Expression, purification, crystallization and preliminary X-ray analysis of response regulator SaeR from Staphylococcus epidermidis

• Main Authors: Yu-Ren Chen, 陳昱任
• Other Authors: Yeh Chen
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/85757854551083531089

碩士 === 弘光科技大學 === 生物科技研究所 === 102 === In Staphylococcus aureus, SaeRS two component system encoded by SaePQRS operon. When SaeS received a specific external stimuli signals, one conserved histidine will be generated for autophosphorylation, and then the phosphoryl group is transferred to a conserved aspartate located on the N-terminal regulator domain of SaeR. However, after phosphorylation the SaeR will be bound onto the DNA through the C-terminal effect domain; the identified binding sequence is GTTAAN6GTTAA, thereby regulates the gene expression of virulence factors, such as α- hemolysin and coagulase. Accordingly, this study expected to understand the interaction between SaeR and DNA as well as study the protein structure of SaeR by the structural biology methods. We made over-expression of SaeR protein and SaeR C-terminal effect domain through E. coli gene expression system and purified them by Ni2+ affinity chromatography and gel-filtration chromatography. After the protein had a high purity, its protein crystals was grown under 4 ℃ by way of sitting vapor diffusion method via many kinds of crystallization conditions. SaeRCTD protein crystal was grown under the crystallization conditions of 0.1 M Bis-Tris pH 5.3, 23% PEG 3350 and 0.2 M MgCl2; its resolution was 2.15 Å through X-ray diffraction; space group was P212121; unit cell dimensions were: a = 34.20 Å and b = 53.78 Å and c = 111.66 Å; an asymmetric unit had two protein molecules. The crystal structure of SaeRCTD proteins was determined by molecular replacement method. We built the SaeRCTD-DNA model for template based on its known structure together with sequence analysis and therefore accordingly believe that these three amino acids, namely, arginine 202, threonine 217 and tryptophan 219, might be primarily involved in DNA binding.