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標題: 大腸桿菌中大腸桿菌素 colicin Ib 之分泌
Secretion of colicin Ib from Escherichia coli
作者: 洪嘉伶
Hung, Chia-Ling
關鍵字: colicin Ib;大腸桿菌素
出版社: 分子生物學研究所
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A fosmid clone from a genomic library of Shigella flexneri that carried the colicin Ib gene (ColIb) was previously isolated. The clone produced an inhibition zone on the E. coli DH5α-containing plate. By comparing the intracellular and extracellular total proteins of an E. coli strain that contained ColIb in a high-copy plasmid with those of the same E. coli strain that contained only the high copy plasmid, I found that extracellular ColIb protein seemed smaller than intracellular ColIb protein. Two recombinant plasmids that carried the promoter and structural gene of col and had a six-histidine tag at the N or C terminus of the encoding ColIb protein were constructed. The tagged protein was induced in E. coli W3110 by mytomycin C treatment, and western analysis indicated that both N and C termini wer intact in the extracellular ColIb protein.

ColIb protein with six-histidine tag at the N terminus under T5 promoter was overproduced in E. coli and purified by affinity chromatography. While using a elution buffer containing 1M imidazole to elute ColIb bound to Ni-NTA agarose column, several proteins were co-eluted with ColIb. LC/MS/MS identified three such proteins, TufA, TufB and TolC. E. coli strains, BW25113 and BW25113 (tolC::km) that carried ColIb gene in plasmid both showed inhibition zone on DH5α-containing plate, but the latter showed bigger inhibition zone, indicating TolC was involved in ColIb secretion.

實驗室曾自一株Shigella flexneri的 fosmid library中,找到了一個具有大腸桿菌素 ColIb基因的clone,並且發現此 clone對E. coli DH5α 可產生抑菌圈。比較實驗室已構築好帶有ColIb基因的high copy 重組質體的E. coli與僅帶有high copy質體的 E. coli 的胞內與胞外蛋白圖譜,發現分泌至胞外的 ColIb 似乎比胞內的 ColIb 小,本研究構築含 ColIb promoter 的 ColIb基因的重組質體並且 ColIb基因所做出的 ColIb 蛋白 N 端或 C 端帶有 6 × His ,發現此兩個質體在 W3110 中皆以 Mitomycin C 誘導 ColIb 蛋白表現,西方雜配證明分泌至胞外的 ColIb,其 N 端和 C 端皆沒有缺失。
將攜帶N 端帶有 6 × His 的 ColIb 質體的 E. coli 以 Ni-NTA agarose column 純化菌體蛋白質中的ColIb-His 蛋白,當利用 elution buffer 沖提出 ColIb 蛋白及以 SDS-PAGE 分析沖提液,發現具有 1M imidazole 的數個與 ColIb 有 interaction 的蛋白。將這些蛋白經質譜鑑定出分別為 TufA、TufB 和 TolC 蛋白。比較攜帶有 ColIb 質體的 BW25113 和 BW25113 (tolC::km) 兩菌對 DH5α 的抑菌作用,發現後者可產生較大的抑菌圈,顯示 TolC 與 ColIb 的分泌有關。
其他識別: U0005-1908201021320900
Appears in Collections:分子生物學研究所

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