表現與純化人類水晶體蛋白

Abstract

本研究是利用載有基因重組質體 (pQE1) 之大腸桿菌 M15/pREP4來生產human γD crystallin (HGDC)，並利用純化方法將HGDC與大腸桿菌自身雜蛋白分離。在第一部份之實驗中我們透過調整大腸桿菌之生長環境，如pH值、誘導劑IPTG的添加量以及添加時機、溶氧量、添加不同碳源、利用乳糖進行誘導、與變更培養液成分之比例等方式，找出最適合大腸桿菌生產HGDC 之環境。於第二部分之實驗中，我們利用acetone沉澱法、親和性吸附層析、及離心過濾等方式，將HGDC與雜蛋白分離，試圖獲得高純度之HGDC溶液，並探討回收率與純化方法之間之關係。 由我們的實驗結果得知以下結論：(1) 培養基起始pH值對於大腸桿菌生長並無明顯之影響，但是以pH值為7時HGDC產量稍高。(2) IPTG的添加為菌體濃度(OD600)達到0.6時，加入0.1mM時最佳。(3) 當溶氧量較高時，細胞生長情況以及HGDC產量均比溶氧量較低時為佳。(4) 添加葡萄糖、半乳糖、及甘油等碳源對於菌體生長以及HGDC的生產均無明顯之提升。(5) 利用乳糖進行誘導時，所獲得之HGDC產量與利用IPTG誘導時產量相近。(6) 透過增加LB培養液中tryptone與yeast extract的濃度，可以有效提升HGDC生產及菌體濃度(OD600)。(7) 透過acetone沉澱法純化時，可獲得接近90%之蛋白質回收率，純度亦可提升兩倍。(8) 利用親和性吸附純化時，可將HGDC純度提高至92%左右，而回收率為17~32%左右。(9) 最後我們利用離心過濾方式，進一步純化親和性吸附產物時，可以得到近乎100%純度之HGDC溶液。

Abstract In this thesis, we used E. coli M15(pREP4), which harbors the plasmid pQE1, to express human γD crystallin (HGDC). After expression, several purification methods were utilized to separate HGDC from the other unwanted proteins. In the first part, we investigated the parameters such as pH of medium, the concentration of IPTG, and the timing of IPTG addition, the dissolved oxygen concentration, the type of carbon source, induction with lactose, the composition of LB medium, and tried to obtain the optimum expression environment for HGDC in E. coli. In the second part, acetone precipitation, affinity chromatography, and ultra-filtration were employed to purify HGDC from the other proteins. In order to obtain the solution with high purity of HGDC, attempts were also made to examine the relationship between the protine yield and the purification methods. Our results can be summarized as follows: (1) The growth of E. coli was not significantly affedcted by the initial pH value of medium. However, in comparison with those obtained at other pH values, a slightly higher yield of HGDC was achieved as the initial pH was set at 7.0. (2) The optimum condition for IPTG addition was found to be OD600 = 0.6 and final concentration = 0.1 mM. (3) Better cell growth and yield of HGDC were observed when the amount of dissolved oxygen was higher. (4) No considerable enhancements in the cell growth and the HGDC production were perceived as glucose, galactose, or glycerol was used as the carbon source. (5) Comparable amouts of HGDC were abtained from the expressions of E. coli with IPTG induction and lactose induction. (6) Through increasing the concentrations of tryptone and yeast extract of LB medium, the productivity of HGDC and the cell density (or OD600) were able to be effectively enhanced. (7) About 90% of the yield and two-fold of the purity were obtained when the protein was purified via acetone precipitation. (8) Using affinity chromatography, the purity and the yield of HGDC were found to be ~92% and ~17-32%, respectively.(9) It was observed that almost 100% pure HGDC solution could be acquired if the product from affinity chromatography was further purified with the ultra-filtration method.