溶菌酶復性程序效率提升之研究

The Improvement of Efficiency in the Lysozyme Renaturation Process

陳勻錡(Yun-Chi Chen)

指導教授 ： 劉懷勝

國立臺灣大學/工學院/化學工程學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.10132

摘要

在蛋白質復性程序中，錯誤的摺疊與聚集體的形成是使復性效率不能提高的主要原因，因此於傳統的直接稀釋復性法中，皆利用大量的復性劑降低蛋白質之濃度並且稀釋變性劑之濃度，以利蛋白質之復性。但於本研究中，利用透析復性法，證實除了錯誤的摺疊與聚集體之外，復性溶液中之還原態二硫代蘇糖醇(dithiothreitol，DTTred)亦是影響蛋白質復性效率的重要因子。有鑑於此，本研究提出一於低稀釋倍率下之高效率復性策略：亦即於低稀釋倍率下，藉由添加氧化態榖胱甘肽(glutathione，GSSG)與DTTred反應，進而提升最終復性產率為傳統操作之13倍，並同時降低操作成本為傳統操作成本之40 %。
此外，本研究亦利用大小排阻層析法(size exclusion chromatography，SEC)可以於復性初期即將影響復性效果之DTTred分離的特性，對高濃度蛋白質進行復性。為了避免蛋白質因濃度過高而於管柱前端形成聚集體影響復性，本研究提出chaperon solvent plug復性方法，並提出最適當之chaperon solvent plug體積及樣品載入之時間，有效地抑制注射閥與管柱間之聚集體形成，避免影響復性效率。並透過對多段式流速復性法及尿素梯度復性法之了解後，提出一較佳之SEC復性策略：在高流速及chaperon solvent plug之保護下，將變性蛋白質載入至系統中，待其進入至SEC管柱後，則於管柱內提供尿素梯度之環境以避免聚集體或錯誤摺疊之蛋白質的形成，並且藉由低流速之操作，增加蛋白質於低尿素濃度區域之滯留時間，使變性蛋白質摺疊回具有活性、正確構型之復性蛋白質。

關鍵字

蛋白質復性 ； 溶菌酶直接稀釋法 ； 大小排阻層析法 ； Chaperon solvent plug

並列摘要

Misfolding and aggregation are the main obstacles of refolding efficiency in protein renaturation process. With the conventional dilution refolding method, large amount of refolding buffer is required to prevent aggregation and to dilute denaturant. In this investigation, via dialysis, besides midfolding and aggregates, reduced dithiothreitol (DTTred) was also proved to be a crucial factor in renaturation. Thus, we proposed a low dilution factor and high efficiency refolding strategy by adding high oxidized glutathione (GSSG) in the refolding buffer to react with carry-over DTTred, resulting in a 13 times of productivity and 60 % reduction of cost, compared to the conventional dilution refolding process.
Because of the benefit of separating DTTred in the beginning of renaturation, lysozyme could be refolded at high concentration by size exclusion chromatography (SEC). We further proposed the optimal volume of chaperon solvent plug and the loading time of sample to prevent aggregations before the column inlet. In addition, we also combined the chaperon solvent plug strategy, urea gradient strategy and step change of mobile phase flowrate strategy to offer an optimal SEC refolding environment. That is, at the beginning of injection, denatured lysozyme was loaded at a high flow rate with chaperon solvent plug. After denatured lyszoyme entered into the column inlet, low flow rate was adjusted to maintain enough retention time of denatured lysozyme in the urea gradient region and low urea concentration region to refold to active lysozyme.

並列關鍵字

Protein renaturation ； Lysozyme ； Direct dilution ； Size exclusion chromatography ； Chaperon solvent plug

參考文獻

Anfinsen C.B., Anson M.L., Edsall J.T., Richards F.M., Advances in protein chemistry, Academic press, New York and London, 1968; 23; 122-127

Anfinsen C.B., Haber E., Sela M., White F. H., Kinetics of formation of native ribonuclease during oxidation of reduced polypeptide chain, Proceedings of the national academy of sciences of the United States of America, 1961; 47; 1309-1314

Anfinsen C.B., Principles that govern folding of protein chains, Science, 1973; 181; 223-230

Armstrong F. B., Biochemistry, Oxford university press, New York, 1989

Baldwin R.L., Rose G.D., Is protein folding hierarchic? II. Folding intermediates and transition states, Trends in biochemical sciences, 1999; 24; 77-83