- 學位論文
連結胜肽對蛋白質表現與純化之影響
Functional Analysis of Linker Peptides on Protein Expression and Purification
若您是本文的作者，可授權文章由華藝線上圖書館中協助推廣。
摘要
在重組蛋白表現及純化的領域裡，親和標記物與融合蛋白結合之技術已被廣泛地應用。通常親和標記物以及欲生產之蛋白的中間需要一段連結胜肽，以利前後兩端的區塊維持各自獨立的結構及功能。為了研究不同的連結胜肽對於蛋白質表現及純化的影響，本研究從根黴菌(Rhizopus oryzae)的葡糖澱粉酵素(glucoamylase, GA)製備重組澱粉吸附區域(starch-binding domain, SBD)，利用其具有兩個澱粉結合位置(starch-binding site, SBS I和SBS II)與生澱粉結合之能力，修飾其成為幫助重組蛋白純化之親和標記物。研究中以綠色螢光蛋白(enhanced green fluorescent protein, eGFP)為標的蛋白，銜接於澱粉吸附區域之N端或C端，兩者之間嵌入不同的連結胜肽，以測試重組融合蛋白之表現及功能。研究顯示大部分融合蛋白能成功表現於大腸桿菌中，各種可溶之重組蛋白均能利用其與澱粉的親合力迅速純化之，回收高純度具有活性的重組融合蛋白。比較連結胜肽位於澱粉吸附區域的N端或C端對於澱粉結合能力之影響，發現後者之融合蛋白與澱粉的最高吸附數值高於前者，可能係因前者的連結胜肽會影響SBS I之澱粉結合能力，所以此澱粉吸附區域較適合放置於連結胜肽及融合蛋白的N端。進一步研究顯示，不論澱粉吸附區域在融合蛋白之N端或C端，都不會影響其解離常數及標的蛋白之活性。此外，本研究發展以攪拌法及澱粉管柱層析法，測試利用玉米澱粉純化蛋白之效率。結果顯示所有的融合蛋白皆可成功純化，雖然利用攪拌法可達到較高的吸附率，但是利用澱粉管柱層析法可獲得較高的回收率，且比攪拌法更省時。本研究發展的技術方便、高效率又經濟，因此值得進一步地發展並延伸其在生技製造上的應用。
並列摘要
The construction of a fusion protein often involves the connecting of two domains or proteins by a peptide linker, which should be designed not to disturb the structures and functions of each domain. Many natural enzymes often consist of two or more distinct modules, which are also connected by a linker peptide. Among which glucoamylase (GA) from Rhizopus oryzae is known to contain a linker of 36 amino acids between an N-terminal starch-binding domain (SBD) and a C-terminal catalytic domain (CD). The SBD possesses a high binding affinity towards raw starch so it was applied in recombinant protein engineering to facilitate the purification of fusion proteins. In this study, enhanced green fluorescent protein (eGFP) was fused with SBD and a few linker variants, including the original linker from R. oryzae GA, in order to investigate the effects of different linker peptides on the expression and purification of fusion proteins, and the SBD and eGFP modules were shuffled between the N- and C- termini while constructing the fusion proteins. According to the results of binding assays, when the linker was located at the N-terminus of the SBD, the starch-binding capacity of SBD was reduced as the Bmax values of the fusion proteins containing an SBD at the C-terminus were lower than those of the fusion proteins containing an SBD at the N-terminus, thus it is more favorable to apply this SBD as an N-terminal affinity tag. On the contrary, the Kd values of all fusion proteins were quite similar, as they were determined to be in the same range (10-6 M level). Furthermore, two purification procedures using corn starch were developed: stirring method and starch column chromatography. Based on the comparison of the purification analyses of both methods, although the starch adsorption rate was promoted in the former, the yield was significantly improved in the latter. As a result, it was indicated that using starch column chromatography was more effective, and it is prospective to be developed into a commercial purification technique due to its convenience, efficiency and economy.
參考文獻
1 Gokhale, R. S. and Khosla, C. (2000) Role of linkers in communication between protein modules. Curr Opin Chem Biol 4, 22-27
2 Cornett, C. A., Fang, T. Y., Reilly, P. J. and Ford, C. (2003) Starch-binding domain shuffling in Aspergillus niger glucoamylase. Protein Eng 16, 521-529
3 Reddy, N. S., Nimmagadda, A. and Rao, K. R. S. S. (2003) An overview of the microbial α-amylase family. African Journal of Biotechnology 2, 645-648
4 Sauer, J., Sigurskjold, B. W., Christensen, U., Frandsen, T. P., Mirgorodskaya, E., Harrison, M., Roepstorff, P. and Svensson, B. (2000) Glucoamylase: structure/function relationships, and protein engineering. Biochim Biophys Acta 1543, 275-293
5 Murphy, M. B. and Doyle, S. A. (2005) High-throughput purification of hexahistidine-tagged proteins expressed in E. coli. Methods Mol Biol 310, 123-130