最近幾年，在生物有機體的基因定序計畫提供很大量的資訊。而這些基因序列資訊結合了最新的分子與結構生物學即成為我們現在所稱的「結構蛋白體學」與「結構基因體學」的概念，即是蛋白質的3D結構決定是以整個基因組為範疇。蛋白質3D結構的相關資訊，其重要性是能提供一些在蛋白質的氨基酸序列中無法看出其功能之相關線索。因此在這個計畫中，我們選擇了在人類中會造成致病因子的幽門螺旋桿菌作為我們整個結構基因體計畫的主角。我們將採用multi-dimensional NMR的技術，來決定新奇（novel）蛋白質在溶液中的的3D結構（分子量小於20千道爾頓）。
幽門螺旋桿菌（Helicobacter pylori, HP）是一個格蘭氏陰性菌、成長慢、具有鞭毛及螺旋外狀的細菌。HP會在人類的胃中生長，幾乎全世界有一半的人口均受此菌感染。而幽門螺旋桿菌感染人類之後的所產生疾病包含有消化性潰瘍、胃黏膜相關的淋巴組織瘤或胃線癌等等。目標基因的篩選主要是能適合在NMR機器上操作，因此有三篩選特性：（1）在PDB的蛋白質資料庫中沒有相似的序列（2）蛋白質結構預測不包含膜間的功能單位(transmembrane domain)（3）單股蛋白質的分子量在20kd以下。

首先，我們挑選了20各基因。這些基因是HP-1144, HP-1215, HP-1423, HP-0892, HP-0496, HP-0495, HP-0274, HP-0902, HP-0199, HP-0817, HP-0222, HP-0385, HP-1049, HP-1219, HP-1324, HP-1425, HP-1492, HP-0032, HP-0458, HP-0187。這些PCR產物均可以利用1-10ng的基因組DNA為模版，在56℃and 53℃這兩個黏合溫度而大量得到。20個基因clone在pGEM-T載體後做DNA定序，所得結果顯示所有的目標基因皆為正確序列（除了HP0199）。接著我們在將這些基因clone至pET-22b(除了HP0199)作蛋白質表現及純化。在這些基因中我先選擇了HP-0222作為之後在CD以及NMR上面的結構探討:由於文獻上指出HP-0222為酸性誘導的蛋白質，因此作了在各種不同pH值下的CD旋光光譜探討；在這些pH值中又進一步的挑選三個pH值做蛋白質熱變性實驗。也因其為酸性誘導的蛋白質，因此我們選擇pH3.5為此蛋白在NMR下的緩衝液條件，以及其化變性探討。NMR部分目前則有one-D與HSQC光譜。

最後，希望我們整個計畫完成不僅可以提供幽門螺旋桿菌的蛋白質結構，也能幫助幽門螺旋桿菌在人類疾病中所扮演的角色以及在蛋白體學上的探討。

In the last few years, genome sequencing projects have produced a wealth of information on the organization of living organisms. The flood of sequence information coupled with recent advances in molecular and structure biology have led to the concept of “structural proteomics” or “structural genomics”, the determination of three-dimensional protein structures on a genome-wide scale. An important use of three-dimensional structural information of proteins is to uncover clues as to a protein’s functions that are not detectable from sequence analysis. In this component project, we will use multi-dimensional NMR techniques to determine the three-dimensional solution structures of novel proteins (molecular weight □ 20 kDa) from the human pathogenic bacteria Helicobacter pylori.
H. pylori is a micro-aerophilic Gram-negative, slow-growing, spiral-shaped and flagellated bacterium that colonizes the stomachs of an estimated half of all humans. (30% of the population in developed and up to 90% of the population in developing countries.) In a subset of humans, H. pylori infection leads to serious disease such as peptic ulcer disease, mucosa-associated lymphoid tissue lymphoma or gastric adenocarcinoma. The target chosen for NMR screening are based on the following characteristics: (1) no sequence homologue in the PDB database (2) no predicted transmembrane domain (3) molecular weight of single chain polypeptide under 20 kDa.

Twenty of the H. pylori genes were chosen for structure studies. These genes are HP-1144, HP-1215, HP-1423, HP-0892, HP-0496, HP-0495, HP-0274, HP-0902, HP-0199, HP-0817, HP-0222, HP-0385, HP-1049, HP-1219, HP-1324, HP-1425, HP-1492, HP-0032, HP-0458, HP-0187.The PCR products could be amplified easily in the presence of 1-10 ng genomic DNA template at annealing temperatures of 56℃and 53℃. No unspecific products were found. These PCR products were sub-cloned into pGEM-T (promega) and checked by DNA sequencing. The results of DNA sequencing showed all of the targets were accurate expect HP0199. We are sub-cloning these targets (expect HP0199) into pET-22b for protein expression and purification. Among these target genes, further structural studies of HP-0222 will be carried out using CD spectrometer and NMR spectrometer. Because HP-0222 is acid-induced protein, so I do the CD experiments in different pHs. And NMR condition that I choose is in pH3.5 buffer condition, one-D and HSQC spectrum are the parts of articles.

The completion of this component project will not only provide novel protein structures of Helicobacter pylori and its associated proteins, but also help the proteomic studies of the roles of Helicobacter pylori in human diseases.

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