大小排阻層析法操作程序對蛋白質復性之影響

Abstract

大小排阻層析法(size exclusion chromatography, SEC)近十年來被應用於蛋白質的復性皆有不錯的效果，其優點在於SEC管柱的多孔性膠體不會與蛋白質起化學反應，純粹只是利用膠體的孔洞，將不同構形的蛋白質分離。雖然以SEC進行蛋白質復性能降低在管柱內聚集體的生成，但SEC蛋白質復性仍需考慮兩個重點：(1)管柱前端聚集；(2)正確雙硫鍵的形成。前者影響到質量回收率，後者影響到活性回收率的優劣。由實驗結果發現，復性緩衝液的尿素濃度對質量回收率有很大的影響，尿素濃度越高，質量回收率越高(6 M以上可達94 %)；而復性緩衝液的pH值對活性回收率相關，在pH約10.5時，活性回收率達最大值(約70 %)。 本論文主要探討SEC蛋白質復性的操作條件，分成三個部分作討論：(1)變性程序的變因對SEC蛋白質復性的影響；(2)復性程序的變因對SEC蛋白質復性的影響；(3)以數學程式來模擬蛋白質於SEC中分離的層析圖。在第一部分中，討論經過不同變性時間以及不同變性蛋白質濃度以SEC進行復性所得的復性效果，實驗結果發現，蛋白質經過變性時間越長，復性活性越差，而蛋白質操作濃度在1~10 g/L時，質量回收率和活性回收率並無太大的差異(分別為70~75 %以及30 %)，當濃度達20 g/L時，質量回收率和活性回收率才有稍微明顯地下降(分別為61 %以及24 %)。第二部分中，我們討論了復性緩衝液的流速、尿素濃度以及pH值對SEC復性效果的影響，低流速可獲得較佳的活性回收率，高流速可獲得較高的質量回收率，而提高尿素濃度可有效抑制聚集體的生成，適當的pH可獲得較高的活性回收率。第三部分我們用Matlab程式模擬蛋白質經SEC分離後的層析圖，已可預測在不同流動相流速下原態蛋白質析出的層析圖。

In recent decades, protein refolding by size exclusion chromatography (SEC) has been proven feasible in terms of its good mass recovery and activity recovery. One of the advantages of SEC is that there is no chemical interaction between protein and the column matrix. In addition, the porous property enables SEC to separate the protein with various conformations. Protein refolding by SEC can minimize the formation of aggregates with careful consideration of the following two important aspects: (1) the formation of aggregates in the inlet tubing prior to column, and (2) the formation of correct disulfide bond in refolding protein. The former is related to protein mass recovery, while the latter is crucial to activity recovery. From the experimental results, it can be concluded that the urea concentration in the refolding buffer has a significant effect on mass recovery as demonstrated by the higher urea concentration yielding higher mass recovery (up to 94 % when urea concentration is higher than 6 M). The pH value of the refolding buffer also affects the recovery of refolded protein significantly with the maximum activity of recovery of 70 % at pH 10.5. This research work mainly focuses on the effects of operating variables of SEC on protein refolding, and can be divided into three parts: (1) the effect of denaturation process on SEC protein refolding; (2) the effect of renaturation process on SEC protein refolding; (3) the simulation of the elution profile of native protein in SEC. In the first part, the influences of the duration of denaturation and the concentration of denatured protein on SEC protein refolding process are discussed. Our experimental results indicated that the activity recovery was decreased with increasing denaturation time. When protein concentrations were within 1 to 10 g/L, the mass recovery ranged from 70% to 75 % and the activity of recovery roughly remained constant (30 %). However, when the concentration of denatured protein reached 20 g/L, the mass recovery and the activity recovery of refolded protein were dropped to 61 % and 24%, respectively. In the second part, low mobile phase velocity led to the better activity recovery, while high velocity resulted in higher mass recovery. It was found that elevating urea concentration in refolding buffer suppress formation of aggregates, and appropriate choice of pH, for example, pH 10.5, enhanced the activity recovery. Finally, a program using the Matlab software was devised to describe the chromatogram of the native protein in the SEC column. Results demonstrated that, via the program we developed, the elution profiles of the native protein under different mobile velocities could be simulated in a reasonable manner.