Xanthomonas campestris pv. Campestris 重金屬耐受性蛋白XC2981的大量表達和結構解析

Abstract

Xanthomonas campestris pv. campestris (Xcc) is a gram-negative bacterium that is phytopathogenic to cruciferous plants and causes worldwide agricultural loss. Therefore, X.campestris pv. campestris str. 17 is chosen for our research. At present, DNA sequences of X.campestris pv. campestris str. 17 have been completely determined. By means of these sequences and through bioinformatics approach, we can discover interesting genes that can be amplified by PCR. Then, optimized conditions could be screened to obtain high purity and large amount of desired proteins. Most importantly, the tertiary structures of the desired proteins could be solved by the powerful X-ray diffraction tool. Several genes correlating with heavy metal ion tolerance were studied in this thesis. XC2981 is deduced as a divalent cation tolerance protein from a bioinformatics study.. From prior molecular genetics studies on the E. coli cutA locus, it was revealed that some mutations in this locus can lead to copper sensitivity due to its increased uptake. Hence CutA is believed to participate in copper ion tolerance in E. coli. We have successfully expressed XC2981 in E. coli in high yield. X-ray diffraction data of native XC2981 crystal diffracted to a resolution of 2.6 A has been collected. However, the phase could not be determined, possibly due to the fact that XC2981 contains only one Se-Met at the N-terminal end. Cocrystallization of XC2981 with heavy metal is another alternative and will be prepared for X-ray diffraction and phase determination. XC3379 is deduced as a ferric uptake regulator (Fur), which can regulate the expression of proteins relative to the uptake of iron by binding with the respective promoter. XC3379 has also been successfully expressed in high yield. However, the best condition for its crystallization is yet to be found.