人類γD型水晶體蛋白表現、純化與聚集行為之研究

Abstract

人類γD型水晶體蛋白(Human γD crystallin，HγDC)具有173個胺基酸，為人類水晶體中的主要成分之一。HγDC由兩個相似且富含β-sheet結構的區域(domain)所構成，與少年性白內障和老年性白分障的發生息息相關。在基因重組HγDC表現與純化的部分，藉由比較不同的起動子、Escherichia coli菌種、培養溫度、isopropyl β-D-thiolgalactorpyranoside(IPTG)的濃度、誘導時的OD600nm與後誘導時間，以找出表現重組蛋白質的E. coli最適系統。於本研究中，成功地純化具有6×Histidine親和性標籤之HγDC；而且由實驗結顯示，最佳表現系統為BL21(DE3)/pEHisHγDC，且其最佳條件為培養溫度為30°C、IPTG為0.5mM、誘導時的OD600nm為2.5與後誘導時間為8小時。藉由圓二色光譜與色胺酸螢光光譜的比較，所獲得的6×His-HγDC蛋白質與文獻值相符合，並且實驗結果亦顯示了具有親和性標籤與不具親和性標籤的HγDC在結構上並無明顯的差異。在BL21(DE3)/pEHisHγDC表現組合中，經過最適合化的培養與純化後，可得的蛋白質產量為~23.3mg/100mL培養液，為文獻值的5~20倍。 在UV-C紫外線照射對HγDC結構影響的部分，利用濁度的量測可發現濁度的增加量和蛋白質的濃度、UV-C照射強度與時間具有正向增加之關係。在照射過程中，明顯地經由肉眼觀察到HγDC形成聚集與產生大量的沉澱。利用胍鹽酸將經UV-C照射後所形成的聚集體進行回溶。比較未經照射之原態HγDC(non-irradiated HγDC)、照射後HγDC之上清液(the supernatant fraction of irradiated HγDC，S)與照射後HγDC沉澱物之回溶液(the re-dissolved precipitated fraction of irradiated HγDC，R)在圓二色光譜、丙烯醯胺螢光淬息與色胺酸螢光光譜量測的結果，發現到三者的HγDC結構特性均有所差異。另外，在實驗的溶液中添加L-cysteine、量測蛋白質硫醇基含量、添加還原劑和不添加還原劑蛋白質電泳之實驗結果，顯示了HγDC照射UV-C過程，雙硫鍵的生成或斷裂在其形成聚集與產生大量沉澱上扮演著重要角色。 在HγDC形成類澱粉纖維的研究部分，利用鹽溶液與酸性條件成功將HγDC誘導生成類澱粉纖維。藉由濁度的量測結果，可知道HγDC在溶液中形成大量聚集體。ANS螢光光譜、色胺酸螢光光譜、丙烯醯胺螢光淬息與圓二色光譜的分析，說明所形成的聚集體呈現結構開展、疏水性區域與色氨酸的裸露、β-sheet結構含量比例下降。藉由ThT螢光光譜的量測，顯示了聚集體具有類澱粉纖維結構的特性，並且利用TEM的觀察與證明聚集體為類澱粉纖維。此外，利用熱誘導開展法的分析，可知HγDC在酸性鹽溶液中，其結構上容易傾向開展、未摺疊之狀態。

Human γD crystallin (HγDC), a 173 amino acid protein, is a primary protein component of the human eye lens. It consists of two highly homologous β-sheet domains and is associated with the development of juvenile and adult-onset cataracts. In the first part of this dissertation, we describe the expression, purification, and characterization of 6×His-HγDC. We optimized recombinant protein expression in an Escherichia coli ex-pression system by investigating factors such as the type of promoter, E. coli strain, culture temperature, isopropyl β-D-thiolgalactorpyranoside (IPTG) concentration, optical density of induction (OD600nm), and duration of IPTG induction. We then purified recombinant HγDC coupled to a six-histidine tag (6×His-HγDC). Our results showed that the optimal system for 6×His-HγDC protein expression was culture of E. coli strain BL21(DE3) bearing the pEHisHγDC plasmid at 30°C and induction with 0.5 mM IPTG once the culture reached an optical density of 2.5 for a period of 8 hr. Circular dichroism spectroscopy and tryptophan fluorescence spectroscopy demonstrated that the structural integrity of the purified 6×His-HγDC protein was identical to that of the previous inves-tigations. We further showed that almost no structural difference was detected between HγDC with and without His-tags. The expression and purification procedure was opti-mized and the resultant final yield (~23.3mg/100mL of culture medium; i.e., ~23.3mg protein produced in 100mL of culture medium) was considerably (~5–20-fold) higher than those reported from previous studies. In the section part of this dissertation, we examined the influence of ultraviolet C irradiation on recombinant human γD crystallin. The turbidity of the HγDC sample so-lution was found to be positively correlated with the concentration of HγDC, UV-C ir-radiance, and UV-C irradiation duration. The HγDC sample solutions became visibly turbid and a noticeable amount of larger protein particles was observed upon prolonged exposure to UV-C irradiation. Circular dichroism spectroscopy, acrylamide fluorescence quenching, and tryptophan fluorescence spectroscopy revealed differences in structures among the non-irradiated HγDC, the supernatant fraction of irradiated HγDC, and the guanidine hydrochloride-re-dissolved precipitated fraction of irradiated HγDC. Our data further suggested that disulfide bond formation and/or cleavage probably play an important role in aggregation and/or precipitation of HγDC induced by UV-C irradiation.

The amyloid fibril-forming propensity of recombinant human γD crystallin was explored in the last part of the dissertation. We successfully induced HγDC into amyloid fibrils in the acidic condition (pH 2.0). We verified the formation of aggregated species in the solutions by observing the increased turbidity. Analyses of ANS fluorescence spectroscopy, circular dichroism spectroscopy, acrylamide fluorescence quenching, and tryptophan fluorescence spectroscopy indicated that the aggregates exhibit features including greater structure unfloding, higher solvent accessibility and reduced β-sheet secondary structure content. Amyloid fibrillogenesis of human γD crystallin in the acidic pH was confirmed by ThT binding assay and transmission electron microscopy analysis. In addition, thermally-induced unfolding analysis allowed us to conclude that the structure of human γD crystallin tends to unfold in acidic condition (pH 2.0).