以梭酸化酵素次單元為標籤之豬胰磷酸脂解酵素的製備與應用

Abstract

哺乳類中分泌型磷酸脂解酵素（14KDa）大致可依其基本結構分成二型：一種為胰臟分泌的pPLA2-I，另一為關節炎型的pPLA2-II。 近來之研究指出pPLA2-I除了消化方面的功能外，亦可透過接受器調控細胞內許多生理功能。例如誘導細胞的增殖，平滑肌收縮，化學增活作用與受精作用等。 PLA2-I接受器的分布已知是在哺乳動物的腎，胃，胰，肺等組織；而接受器分子量從180-200KDa不等。至於其他組織是否也有此胰磷酸脂解酵素接受器的存在目前並不是很清楚。 為了進一步研究此類接受器在各類組織，細胞，尤其是癌細胞中之分佈，我們建立一個非放射性且簡易的檢測方法，我們採用大腸桿菌醯基合成酵素中的一個次單元：含生物素甲基傳遞蛋白質的(amino acid:80-157) BCCP；(Biotinylated carboxylase carrier protein)作為胰磷酸脂解酵素的標記。我們建構了數個接合蛋白質的表現型質體，為了找出最適於作為標記的生物素接和區域，我們對甲基傳遞蛋白質以分子生物技術作不同程度的截斷（分別為BCCP80-136，BCCP80-144，BCCP80-151及BCCP80-156），並將這些接合蛋白置於大腸桿菌BL21（DE3）中進行大量表現，針對這些蛋白質我們將測試其活性,生物素標記的比率及對胰磷酸脂解酵素受體的親和力。

Mammalian Secretory phospholipases A2(sPLA2,14KDa) are classified into two types : the pancreatic group I(PLA2-I) and the arthristic group II, on the basis of their primary structure. Recent reports indicate that PLA2-I, in addition to its digestive function, has receptors-mediated effects. For instance, PLA2-I,via its receptor, can induce cell proliferation, airway and vascular smooth muscle contraction, chemokinesis and fertilization. PLA2-I receptor is present in various kinds of organs, including mammalian stomach, pancreas, spleen and lung . These receptors with molecular masses of 180-200KDa, have been identified in rabbit, rat, mouse, human and bovine tissues. To elucidate the distribution of PLA2-I receptor in different tissues and cells , especially in cancer cell ,we developed a simple non-radioisotope approach. A truncated Biotin carboxylase carrier protein (BCCPt , a.a.: 80-157) was used as a tag to label PLA2-I by fusing both porcine PLA2-I gene with truncated BCCP fragment cDNA. To find out the smallest domain of BCCPt that can be still recognized and labeled by biotin ligase , we prepared various pPLA2-I-BCCPt fusion constructs containing different length of BCCP domain, including BCCPt(80-136), BCCPt(80-144), BCCPt(80-151) and BCCPt(80-156). These expression constructs were over-expressed in E.Coli strain BL21(DE3). The result showed that PLA2-I-BCCPt could be over expressed in BL21(DE3), although in a form of inclusion body. The biotinylation of all four fusion proteins were observed as detected by Streptavidin-conjugated HRP followed by TMS colorimeteric development.