以大小排阻層析法進行蛋白質復性之研究

Abstract

在蛋白質復性程序中，形成錯誤的摺疊與聚集體是降低復性效率的主要原因。實驗結果證實，注射閥與管柱間所形成的聚集反應嚴重阻礙SEC(size exclusion chromatography)的復性效率，因此，本研究即在探討應如何抑制注射閥與管柱間的聚集反應以提昇復性效率，並在不同復性劑組成與稀釋倍率條件下相較於直接稀釋法所得復性結果，以嘗試了解管柱內的多孔材質對於輔助蛋白質摺疊之機制。 本研究提出兩種方法來抑制注射閥與管柱間的聚集反應，即Chaperone solvent plug復性法與流動相兩段速復性法。Chaperone solvent plug 復性法是利用高濃度的變性劑提升失活蛋白質在遷移過程時的穩定性，藉以抑制聚集體的形成; 流動相兩段速復性法則是先採取高初始流速方式以抑制管柱前端所生成的聚集反應，待失活蛋白質進入管柱後則採取低流速方式以提供蛋白質進行正確摺疊的所需時間; 因此，活性回收率能否提升將取決於蛋白質在管柱內的滯留時間。此外，將流動相兩段速結合chaperone solvent plug復性法將可完全取得100%的質量與活性回收率。 為能了解注射閥與管柱間如何進行聚集反應，本研究亦探討不同規格樣品環(改變管徑或管長)對於聚集體的形成機制與復性效率之影響，並獲得高注入體積與低進料濃度為提升復性效率的最佳注入方式。 此外，亦採用氧化還原復性劑的梯度方式動態調控雙硫鍵的形成，並在不同復性劑組成條件下，比較isocratic SEC復性法與稀釋法所得復性結果，發現低稀釋倍率的復性方式可突顯SEC的多孔材質具有輔助或催化蛋白質摺疊之能力，尤其在缺乏GSH(reduced glutathione)或GSSG(Oxidized glutathione)的復性環境下，可取代部份GSH與GSSG在氧化還原雙硫鍵之角色。

The formation of incorrectly folded protein, in particular aggregates, was recognized as the hindrance of good yield in refolding processes. Results from our study have shown that the formation of aggregates between the injection valve and column inlet was found to strongly hamper (adversely affect) the efficiency of lysozyme refolding in size-exclusion chromatography (SEC). To enhance the performance of protein refolding, our studies were directed toward seeking out methods or means to reduce the aggregate production during the SEC refolding. Furthermore, we compared the refolding performances between the direct dilution and SEC refolding methods under different constituents of refolding buffer or dilution factors, and then examined how the protein refolding was assisted via porous matrix in SEC column. In this study, chaperone solvent plug strategy and step change of mobile phase flowrate strategy were developed to overcome the aggregate formation between the injector and column inlet. For the former one, the denatured protein was escorted from the injector into the column by a solvent plug that could inhibit aggregate formation or stabilize the denatured protein. As to the latter one, a higher flowrate of mobile phase should be applied to reduce the traveling time of denatured protein from injector to column inlet. After the denatured protein penetrated into the column, a lower flowrate should be used to allow enough time for protein to refold. Enough residence time inside the column was identified to be the key for proper protein refolding. Then combining this method with chaperone solvent plug strategy could achieve complete recovery of denatured proteins, both mass and activity recoveries. Furthermore, we also investigated how aggregate formation as well as renaturation yield varied with the tubing dimension (diameter or length) of sample loop in the SEC refolding process. It was found that a sample with large volume and low concentration was preferable for refolding process. In addition, via a gradient of redox buffers through the SEC, oxidative refolding was achieved under kinetic control the disulfide bond formation. Employing the renaturation buffer system with different redox constituents and comparison with batch dilution, we demonstrated that the porous matrix packing in SEC could assist protein refolding in the absence of reduced and oxidized glutathione (GSH/GSSG).