解整合素與金屬蛋白酶家族之蛋白質表現、純化與復性分析的研究

Abstract

解整合素與金屬蛋白酶(A Disintegrin And Metalloproteinase, ADAM)具有細胞黏附活性和水解活性，可切割細胞膜外區域的穿膜蛋白，在解整合素與金屬蛋白酶家族中，解整合素與金屬蛋白酶12 (ADAM12)被指出與腫瘤細胞的轉移或形成有關，而解整合素與金屬蛋白酶29 (ADAM29)也與癌症有關，因此ADAM12和ADAM29可以作為癌症免疫治療之標的。 本研究使用三種解整合素與金屬蛋白酶(小鼠的ADAM12、人類的ADAM12和小鼠的ADAM29)作為重組蛋白質的來源，並選擇其中具有受質辨識功能的多半胱胺酸區域(Cystein-rich domain)作為目標，在異體宿主(Heterologous host)大腸桿菌中進行蛋白質表達與純化。本研究已成功的在大腸桿菌中表達出目標蛋白質，並有足量的可溶蛋白質以進行純化。除了利用金屬親和層析來進行蛋白質純化外，也利用管柱內復性的方式，得到更大量純化之目標蛋白質，最後以西方墨點法(Western blotting)的方式檢測目標蛋白質，其結果支持本研究所得之純化的蛋白質，本研究有助於促進癌症疫苗在治療上的應用與發展。

ADAM (A Disintegrin And Metalloproteinase) has been reported to play a role in cell adhesion and shedding that are responsible for the proteolytic degradation of the extracellular matrix (ECM). Within the ADAM family, ADAM12 and ADAM29 have been implicated in tumor cell lines. These features have made ADAM12 and ADAM29 a possible, suitable target for immunotherapy by vaccine treatment. This study has selected the cystein-rich domains of ADAM12 and ADAM29 as target antigens. Three sources of recombinant cystein-rich domains of ADAM were used in this study, i.e. mice ADAM12, human ADAM12 and mice ADAM29. A heterologous expression system for the production of ADAM12 and ADAM29 proteins was used. The results showed that we have successfully expressed cystein-richs domain of ADAM12 and ADAM29 proteins in E. coli, leading to production of sufficient amount of soluble proteins for purification. Subsequently, metal affinity chromatography was used for protein purification. Alternatively, on-column refolding was also used to get more target proteins. The identity of these proteins was verified by use of western blotting method. The results obtained from this study can be helpful for promoting the development of cancer vaccine for possible therapeutic applications.