Chromatography analisis of the interaction between XpsG and XpsH of Xanthomonas campestris pv. campestris

• Main Authors: Feng-Chang Wu, 吳逢昌
• Other Authors: Ling-Yun Chen
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/13846511345128816583

碩士 === 中山醫學院 === 生物化學研究所 === 88 === Abstract In Gram-negative bacteria, Type II extracellular protein secretory system is comprised of at least 12 to 13 gene products. Among those proteins, GspG, H, I, and J proteins are referred as pseudopilin. The entire structure of pili, which composed of GspG, H, I, and J proteins, however has not yet been identified. The specific aim of this research is focusing on the possibility of formation of pili structure in the extracellular protein secretory system by using antibodies against XpsG, H, and I from Xanthomonas campestris, respectively. Subcellular distribution analysis showed that both XpsG and XpsH proteins were existed in soluble and insoluble fraction. Gel filtration analysis showed that the molecular weight of XpsG membrane form was about 30-50 kDa under the treatment of 0.5% DOC；while it was about 200 kDa under the treatment of 2% Triton X-100. The molecular weight of XpsG soluble form was over 600 kDa, but while treated with DOC or Triton X-100 the multimer was dissociated. DOC caused the multimer form dissociated to 30-50 kDa, Triton X-100 caused the multimer form dissociated to 200 kDa, which similar to XpsG membrane protein. Both chromatogram of membrane and soluble form of XpsG were unchanged whether the XpsH protein existed or not. In XpsH protein, the molecular weight of XpsH membrane form was about 40-60 kDa under the treatment of DOC, slightly larger than XpsG membrane protein. While treated with Triton X-100, the molecular weight of XpsH was over 600 kDa. These results suggested that membrane-bound XpsH protein could be a macromolecular form, which would be completely destroyed by DOC, but partly destroyed by Triton X-100. In XpsH soluble form, its molecular weight was over 600 kDa based upon gel-filtration chromatography. This multimer form could disassociate into 400 kDa and 70 kDa when equilibration buffer contained DOC. When equilibration buffer contained Triton X-100, this multimer disassociated into 200 kDa. Since the molecular weight of both membrane and soluble XpsH proteins are different from each other under the treatment of DOC and Triton X-100, it implied that the structure of both forms of XpsH protein is different from each other. In anion-exchange chromatography analysis, it showed that both soluble XpsG and XpsH proteins are cofractionated in the elution profile. Site-directed mutagenesis was also performed on XpsG protein in this study. Conservative amino acids D70, D121, D128 and D139 of XpsG protein have been mutated to Glu and only D139N mutant protein showed the lost of its function. This function lost was due to the degradion of mutated protein itself. The effect of single amino acid change, which caused the tremendous effect of protein stability, remains unclear.