人類凝血酶調節素的表現、純化、結晶與小角度X光散射實驗分析

Abstract

人類凝血酶調節素（Thrombomodulin, TM）是內皮細胞表面的穿膜醣蛋白。此蛋白質由N端至C端可分成五個不同的結構區域：Lectin like domain、EGF-like domain、serine/threonine-rich domain、transmembrane domain以及cytoplasmic domain。人類凝血酶調節素的每個結構區域參與了各種生化反應，如血液凝結、血纖維蛋白溶解、免疫反應、細胞附著及增生。 人類凝血酶調節素參與的各種生化功能和其結構有關，為了探討人類凝血酶調節素的結構區域如何參與各種生化反應，我們希望藉由蛋白質結晶繞射實驗來解出其結構，利用詳細的結構來探討TM的功能及生化反應機制。我們使用酵母菌表現系統表現了參與特殊功能的TM片段：TMD-1；包含了c-type Lectin like domain、TMD-23；包含了EGF-like domain以及serine/threonine-rich domain。 酵母菌表現的TMD-1以及TMD-23皆有醣化現象，由SDS-PAGE發現TMD-1有較嚴重不均勻的醣化，可能會影響蛋白結晶，因此我們另外使用大腸桿菌表現系統來表現TMD-1。酵母菌表現的TMD-23經由鎳離子螯合親合性色層分析法（Ni-chelating sepharose fast flow）純化，進行蛋白質養晶實驗。酵母菌表現的TMD-23（濃度為12 mg/mL，25 mg/mL，36 mg/mL）、利用水蒸汽擴散法（Vaper diffusion）進行蛋白質結晶實驗。在嘗試數種結晶試劑篩選之後，結果發現沒有好的蛋白質結晶生成。另外藉由大腸桿菌表現的TMD-1幾乎都是包函體，我們利用on-column refolding的方法進行蛋白質復性。我們將復性後的TMD-1（濃度為5 mg/mL）進行蛋白質養晶實驗，但TMD-1是在結晶過程中容易沉澱，不易形成結晶。 此外由於近年來超巨分子以及複合體的研究需求，CryoEM 以及 SAXS （small angle X-ray scattering; 小角度X光散射）等的低解析度結構資訊與高解析度結構資訊的整合，可以使我們的X光單晶晶體繞射及NMR結果可以更快的經由像拼圖的方式，得到超巨分子以及複合體的結構資訊。因此我們也將使用小角度X光散射的方法來觀察TM在水溶液裡的低解析度結構。我們進行TMD-23的小角度X光散射實驗（Small angle X-ray scattering），配合電腦軟體以dimer以及monomer模擬TMD-23在水溶液中和Ca2+結合前後的結構變化，而此結果仍須在未來做進一步的研究。

Thrombomodulin (TM) is a multifunction glycoprotein expressed on the epithelial cell surface. This glycoprotein is structurally organized into 5 distinct domains. From the N-terminus to C-terminus, TM has an N-terminus lectin domain, an EGF-like repeats, and a serine/threonine-rich region which were on the extra-membrane, a transmembrane domain and a short cytoplasmic tail on the intra-membrane. Each of the distinct domains has different biological functions that impact on coagulation, fibrinolysis, inflammation, cell Adhesion, and cell proliferation. To elucidate how the single molecule can play diversely important role by each distinct domain, we expect to obtain the crystal structure of each TM domains. By TM structure, the functions and biochemical mechanisms will be clearly analysed and annotated. Here, we use the Pichia Pastoris expression system to express different functional domains of TM: the TMD-1 construction contains the C-type lectin domain, the TMD-23 construction contains the EGF-like repeats and the serine/threonine-rich region. Both Pichia Pastoris expressed TMD-1 and TMD-23 were glycosylated proteins. The unequal glycosylation of TMD-1 may influence protein crystallization, therefore we use E.coli to express TMD-1. The Pichia Pastoris expressed TMD-23 was purified through Nickel affinity column. By using vapor diffusion method, we test some crystallization conditions of TMD-23 (12 mg/mL，25 mg/mL，36 mg/mL). Unfortunately, there is no good protein crystal harvested. The expression of TMD-1 in E. coli results in the formation of inclusion bodies. We use the on-column refolding to obtain the soluble form of TMD-1 and test some crystallization conditions of soluble TMD-1 (5 mg/mL), but the TMD-1 is instable, it often aggregated itself. In addition, due to in recent years the large macromolecule and protein complex research rised, The structural information of CryoEM and SAXS (Small Angle X-ray Scattering) can combined with the structure of crystal diffraction or NMR. We will also use small angle x-ray scattering for observing the low resolution structure of TM in the aqueous solution. We use small angle X-ray Scattering to model the monomer and dimmer shape of TMD-23 with/with out the binding of Ca2+. The model results need to be investigated in the future.