小鼠肝臟醯亞胺水解酵素之選殖、表現及再摺疊

Abstract

醯亞胺水解酵素是肝臟中的解毒酵素，其已知生理功能為代謝嘌呤途徑中的第二個酵素。本實驗主要的目的是希望能在大腸桿菌中表現小鼠醯亞胺水解酵素，以簡化純化步驟及增加產率。首先，我們以未定出醯亞胺水解酵素核酸序列的小鼠為實驗材料，從鼠肝中分離出所有的核醣核酸後，以反轉錄聚合酶連鎖反應及聚合酶連鎖反應合成第一股及第二股的互補去氧核醣核酸，接到轉殖載體，轉形至大腸桿菌DH5α中大量複製後，定出小鼠醯亞胺水解酵素核酸序列，轉錄區域為1560個鹼基，和大鼠的醯亞胺水解酵素相同性為92%，和人類的醯亞胺水解酵素相同性為86%，確定所選殖到的小鼠基因為醯亞胺水解酵素，轉譯為氨基酸序列共519個氨基酸組成，相同性又再度升高，和大鼠的相同性為94%，和人類的相同性為88%。之後我們將質體分離出，利用限制酶作用切下醯亞胺水解酵素基因片段，接到pET23a，轉形至大腸桿菌BL21-RIL (DE3)中，使小鼠肝臟中的醯亞胺水解酵素基因在大腸桿菌中表現，產生醯亞胺水解酵素，由於所生成的蛋白質為包含體 (inclusion bodies)，利用降低IPTG濃度、降低誘導溫度、更換培養液為M9，仍形成了包含體。之後並進行了再摺疊 (refolding) 的步驟，但仍然無法得到有活性的酵素。

Imidase is an antidotal enzyme from mammalian liver, and it is the second enzyme involved in pyrimidine metabolism pathway. We clone and sequence an imidase cDNA from mouse liver. The coding region of mouse liver imidase cDNA is 1560bp. The identity of imidase DNA of mouse is 92% and 86% between rat and human, respectively. Translation of DNA sequences indicates 519 amino acids. The identity of amino acid sequence of mouse is 94% with rat and 88% with human. The imidase cDNA is subcloned to expression vector pET23a and transformed to Escherichia coli BL21-RIL (DE3). The production of recombinant imidase from Escherichia coli is found to cause protein aggregation. We reduce the concentration of IPTG, lower temperature and change medium of the cell culture in a variety of experiments. There is no significant amount of soluble protein according to SDS-PAGE and no imidase activity is detected. We used 6M urea and 6M guanidine-HCl to dissolved the inclusion bodies. The refolding process to reactive imidase activity is not successful.