結核桿菌PONA1轉醣酶之純化與結晶

結核桿菌(Mycobacterium tuberculosis)是引起肺結核(tuberculosis)的主要致病菌。據世界衛生組織(World Health Organization；WHO)統計，全球約有三分之一的人口感染過結核桿菌，且每年約有一百萬人因肺結核而死亡。廣泛使用抗生素的結果導致結核桿菌抗藥性菌株的產生，而這在結核病的治療上是一項相當大的難題。結核桿菌中有兩個大分子量的Class A青黴素結合蛋白(high molecular mass class A penicillin-binding proteins；HMM Class A PBPs)，分別為PONA1和PONA2。兩者皆為膜蛋白且同時具有轉胜肽酶 (transpeptidase；TP)與轉醣基酶(transglycosylase；TG)的酵素活性，這些酵素在細菌細胞壁胜肽聚醣(peptidoglycan)的合成上扮演著很重要的角色。因此，能夠抑制這兩種活性的藥物，理論上能夠有效對抗結核桿菌。但目前臨床上用於治療肺結核的第一線與第二線抗生素並沒有以此兩種酵素活性作為標的。為此，結核桿菌HMM Class A PBPs的結構，在未來肺結核藥物設計上是一項極具價值的研究。本篇論文研究中，結核桿菌H37Rv株PONA1膜蛋白及其不同長度的片段以大腸桿菌(E. coli)為表現系統並利用介面活性劑進行膜蛋白萃取，將萃取出來的蛋白質以鎳離子螯合樹脂(Ni-NTA resin)純化，粗純化的蛋白質進一步以尺寸排除層析法(size exclusive chromatography)分離單元體及二元體。此外，同一製程的蛋白質同時利用NBD-Lipid II進行TG活性測試與利用SPR測量蛋白與moenomycin的結合能力，以確保結晶的蛋白具備活性。實驗中，我們嘗試了各種不同的介面活性劑，進行結晶條件的測試，以期取得moenomycin 與蛋白結合的晶體，做為將來藥物開發的依據。

關鍵字

結核桿菌PONA1 ；膜蛋白；轉醣酶蛋白質結晶

並列摘要

Mycobacterium tuberculosis (Mtb) is the major cause of tuberculosis. According to the statics of World Health Organization (WHO), about one-third population in the world was infected by M.tuberculosis. Almost one million people die of tuberculosis each year. The abuse of antibiotics results in drug resistance strains. The drug resistance makes a significant barrier to the treatment of tuberculosis. Two high molecular mass class A penicillin-binding proteins, PONA1 and PONA2, (HMM Class A PBPs) are cloned from genome DNA of Mtb H37Rv strain. Mtb H37Rv PONA1 and PONA2 play important roles in the biosynthesis of peptidoglycan. Both of them are membrane proteins with two functional domains, transpeptidase (TP) and transglycosylase (TG). Therefore, the drugs can inhibit those enzymes, could theoretically against tuberculosis effectively. Until now, the first-line and second-line anti-tuberculosis drugs are not specific to these enzymes. For this reason, to solve the HMM Class A PBPs protein structure of Mtb is a quite valuable research project for the structure-based drug design. In this study, we express the PONA1 and various truncated of PONA1 from Mtb H37Rv with N-terminal His-tag in E. coli expression system. The protein is purified with Ni-NTA and size exclusive chromatography in the present of different detergents. Moreover, the PONA1 and various truncated of PONA1 are examined TG activity by NBD-Lipid II and tested binding affinities between moenomycin and protein by SPR in order to make sure the material proteins are functional. Finally, the complex of purified proteins and moenomycin were crystallized in the present of difference detergents by crystallized screening kits and hope to get the structure for the structure-based drug design.

並列關鍵字

Mycobacterium tuberculosis ； PONA1 ； transglycosylase ； membrane protein ； crystallization

參考文獻

1. WHO Tuberculosis Factsheet, World Health Organization, March 2010

2. Jasmer, R.M., P. Nahid, and P.C. Hopewell, Clinical practice. Latent tuberculosis infection. N Engl J Med, 2002. 347(23): p. 1860-6.

4. Kittisupamongkol, W., Extrapulmonary tuberculosis. South Med J, 2009. 102(7): p. 776.

5. Goldrick, B.A., Once dismissed, still rampant: tuberculosis, the second deadliest infectious disease worldwide. Am J Nurs, 2004. 104(9): p. 68-70.

6. Mannepalli, S., et al., Mycobacterium tuberculosis osteomyelitis in a patient with human immunodeficiency virus/acquired immunodeficiency syndrome (HIV/AIDS): a case report. Cases J. 3: p. 67.

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結核桿菌PONA1轉醣酶之純化與結晶

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