化膿性鏈球菌 (Streptococcus pyogenes)，俗稱A 群鏈球菌 (簡稱GAS)，是人類常見致病菌之一。A 群鏈球菌會導致人類產生許多的疾病，其中包含輕微症狀的咽喉炎、膿皰疹，嚴重的則有猩紅熱、蜂窩性組織炎、毒性休克症候群、壞死性肌膜炎...等。近年來許多 GAS 菌株的基因體已經被定序出來，其中我們所研究的 M1 菌株大約有 2000 多個基因，當中有 33 % 的基因功能是未知的，特別的是這些未知功能的基因，大部分是源自於 Bacteriophage 或 transposon 嵌入菌體所留下的基因。過去的文獻指出，噬菌體感染細菌後，可能會增加細菌體本身的致病力，如增加菌體入侵、附著與生長的能力；抵抗免疫系統或抗生素的攻擊；或是轉譯出來的蛋白具有毒力。而這次我們所研究的主題是要探討一種稱為 SPy0985的噬菌體關聯性的蛋白 (phage-associated hypothetical protein)，蛋白由88個胺基酸組成，其中第49至第75的胺基酸序列經由軟體預測分析結果認為具有超抗原的活性(superantigen activity)。目前已利用大腸桿菌 (E. coli BL21) 成功表現SPy0985蛋白並予以純化，平均產率約為 30 mg/L。並且我們找到 SPy0985在 50 mM arginine and glutamate (R+E) buffer 條件下，會有結構的形成以及減少蛋白的聚集物 (aggregation)；利用膠體色層分析法確認蛋白的分子量，發現不論在NMR buffer條件或是生理條件的環境中，SPy0985形狀近似二聚體 (dimer)；週邊血液單核細胞增生測定 (PBM cell proliferation assay) 顯示其可能具有超抗原的活性。SPy0985的結構以及動力學結果顯示，蛋白的N端以及C端有較高的NOE數值，其C端有形成兩個互相作用的α-helices，並且在N端由F8、Y17、Y23、L31以及Y36等疏水性胺基酸交互堆疊；在G39-D51區域有較低的NOE數值，這表示此區域有flexible loop的形成；有趣的是，在SPy0985的2D 1H-15N HSQC圖譜，發現蛋白N端以及loop區域的胺基酸K3-T46具有兩個訊號(resonances)，這表示此區域有結構上的變換 (conformational change)；SPy0985在動力學NOE數值為0.58，顯示SPy0985整體較為flexible，其R2/R1數值表示SPy0985在NMR buffer條件下為單聚體 (monomer)。目前的研究結果，我們對於SPy0985蛋白結構以及功能的細節有待進一步的探討。

Streptococcus pyogenes (group A Streptococcus [GAS]) is one of the most common human bacterial pathogens. GAS causes a variety of human diseases, including pharyngitis, impetigo, scarlet fever, cellulitis, toxic shock syndrome, necrotizing fasciitis. The genomes of many GAS strains have been sequenced, and they contained ~2000 genes. However, the functions of ~33% GAS genes are still unknown. In particular, most of these unknown genes are bacteriophage and transposon genes. It has been known that phage infection can enhance bacterial virulence, including bacterial adhesion, colonization, invasion; resistance to immune defenses; exotoxin production; sensitivity to antibiotics; and transmissibility among humans. In this study we identified a phage-associated hypothetical protein, SPy0985, which contains 88 amino acids residues with a superantigen signature sequence at the position of 49-75. SPy0985 was successfully expressed in E. coli and purified to homogeneity with a yield of 30 mg/L. We also found that of SPy0985 in the presence of 50 mM of arginine and glutamate (R+E) was properly folded with less aggregation. Molecular weight determination by size-exclusion chromatography analysis showed that SPy0985 existed as a shape of dimeric size under R+E and physiological conditions. The result of peripheral blood mononuclear cell assay showed that SPy0985 was a superantigen. NMR analysis showed that SPy0985 exhibited stable structures at N- and C-terminal regions with positive NOE and consisted of two α-helices packed against each other at C-terminus. It also formed a hydrophobic core packed by F8, Y17, Y23, L31 and Y36 at N terminal region. NMR relaxation studied showed that residues G39-D51 exhibited low NOE values, indicating the formation of a flexible loop in this region. Interestingly, the N-terminus and the loop ranging from K3 to T46 exhibited two resonances in 2D 1H-15N HSQC spectrum, indicating a chemical exchange process found from this region. The average NOE value of Spy0985 was 0.58, showing that SPy0985 was highly flexible. The R2/R1 ratio indicated that SPy0985 existed as a monomer. The underlying functional and structural details of SPy0985 remain to be elucidated.

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王志傑 Streptopain 核磁共振之研究：C 端及催化圈環對於與抑制劑結合及蛋白酶活性所扮演的角色 (2006)
陳俊良 探討化膿性鏈球菌毒力因子 SPE B及SPy0985之核磁共振結構 (2008)