幾丁質結合蛋白的應用

Abstract

CBP21是一種幾丁質結合蛋白（chitin binding protein），來自於沙雷氏菌Serratia marcescens（革蘭氏陰性桿菌），當將 S. marcescens 培養於以幾丁質為碳源的培養液中，CBP21將隨著其他幾丁質酵素的分泌，而同時被分泌出來。CBP21由197個胺基酸組成，其N-端27個胺基酸為訊息胜肽（signal peptide）。近年來，研究人員發現CBP21具有破壞聚乙醯胺基葡糖直鏈結構的作用，憑藉CBP21的幫助，幾丁質酵素可以更有效地水解聚乙醯胺基葡糖。而在先期試驗中知道CBP21在pH 8.0條件下能與幾丁質結合，而在pH<7條件下便可以洗脫。因此，應可將其建構成純化蛋白質的工具或作為實行酵素固定化之用。利用此特性，吾人應用分生剪接（PCR）技術，將CBP21基因及在其下游續接連接肽（linker）、蛋白酶切位點（protease cut site）及限制酵素多重切點（MCS）等DNA序列，建構於pRSET A表達載體上，再於限制酵素MCS位置分別嵌入數種糖類水解酵素之基因，並將之轉殖入大腸桿菌表現細胞BL21（DE3）中進行表現。利用自製幾丁質細粒管柱進行純化，已成功純化得純度90 % 以上且回收率達40 % 以上的CBP21融合的標的蛋白質。

CBP21 is a chitin-binding protein from Serratia marcescens, a Gram-negative soil bacterium. When S. marcescens is grown in the presence of chitin as a carbon source, CBP21 (197 amino acids, including a 27-residue leader peptide) is one of the major proteins produced and secreted along with other chitinases. In recent years, researchers have found that the non-catalytic CBP21 can bind to the insoluble crystalline substrate and lead to structural change of substrate. Consequently, CBP21 can increase the substrate accessibility and strongly promote the hydrolysis of crystalline β-chitin by other chitinases. Previous study also showed that CBP21 can bind on chitin at a pH value around 8.0 and can be eluted in acid condition (pH < 7). This feature is potentially useful for establishing a system for protein purification and for serving as a tool in enzyme immobilization. Through the application of PCR amplification, a DNA fragment containing the CBP21 gene, a downstream peptide-linker and a genease cut site was obtained. The fragment was successfully inserted into the pRSET_A expression vector. Several genes of glycoside hydrolases, including chitosanase, chitinase, and laminaripentaose-producing β-1,3-glucanase (LPHase), were inserted into the multiple cloning site of the constructed plasmid and further transformed into the E. coli BL21 (DE3) for fusion-protein expression. The capability of this system was then evaluated using β-form chitin as matrix for affinity-column purification. Results showed that proteins with high purity (> 90% homogeneity for all cases) were easily obtained. The recovery yield was more than 40 percent.