摘要
本文主要考察甜菜碱和海藻糖两种渗透剂对蛋白质稳定性、蛋白质折叠复性和蛋白质分子相互作用的影响,以阐明这些渗透剂在蛋白质折叠复性中的作用,进而应用于辅助基因重组包含体蛋白质的复性。
首先,以溶菌酶为模型蛋白质,应用荧光光谱考察了化学变性剂盐酸胍作用下,甜菜碱或海藻糖对天然态溶菌酶去折叠过程中内源荧光的影响。以线性外推模型得到了溶菌酶去折叠过程的热力学参数。结果表明,甜菜碱或海藻糖均可以提高盐酸胍诱导溶菌酶去折叠过程的自由能变化,增大天然态溶菌酶的稳定性。
第二,以完全变性还原溶菌酶为模型蛋白质,考察了添加甜菜碱或海藻糖对其复性的影响。对于甜菜碱而言,随添加浓度逐步升高至2 mol·L-1,溶菌酶的复性收率也逐步升高;对于海藻糖而言,存在最佳的浓度0.2 mol·L-1使得溶菌酶的复性收率最高。以蛋白质折叠与聚集的三级竞争反应动力学模型分析了变性还原溶菌酶复性过程的宏观动力学行为。甜菜碱可以促进变性溶菌酶折叠而促进复性。由于海藻糖对变性态蛋白质具有稳定作用,较低浓度的海藻糖(<0.2 mol·L-1)可以抑制变性态溶菌酶的聚集,提高复性收率;但较高浓度的海藻糖(>0.2 mol·L-1)会导致溶菌酶的折叠减慢,从而使复性收率降低。
第三,为了分析渗透剂在蛋白质复性过程中对蛋白质分子间相互作用的影响,利用自相互作用色谱分析了两种pH下,氯化钠和盐酸胍体系中甜菜碱、海藻糖和精氨酸对溶菌酶分子相互作用的影响,得到了不同条件下溶菌酶的第二维里系数。所有盐酸胍浓度下,随甜菜碱或海藻糖浓度的逐渐增大,溶菌酶的第二维里系数均逐渐增大。但溶液中不含盐酸胍或含有0.05 mol·L-1的盐酸胍时,精氨酸浓度的增大会导致溶菌酶的第二维里系数降低;当盐酸胍浓度超过0.5 mol·L-1时,精氨酸浓度增大则会导致溶菌酶的第二维里系数略有升高。这一结果表明渗透剂和精氨酸对蛋白质分子之间相互作用的影响不同。
最后,通过培养基因工程菌得到了以包含体形式表达的大肠杆菌苹果酸脱氢酶,作为模型蛋白质考察寡聚蛋白质及包含体的复性过程。结果显示,随甜菜碱浓度的升高,复性后苹果酸脱氢酶的比活增大;而海藻糖则出现最佳浓度(0.5 mol·L-1)使得复性效果达到最高;在盐酸胍浓度较高(0.4 mol·L-1)的复性体系中,加入合适浓度的甜菜碱或海藻糖仍可有效促进苹果酸脱氢酶的复性。
This thesis is concerned with an extensive study of protein stability, folding and molecular interaction in the presence of osmolytes, betaine and trehalose. The purpose of the research is to give clearer insight into the role of the osmolytes in the refolding or renaturation of proteins to be recovered from inclusion bodies.
At first, guanidinium chloride-induced unfolding of lysozyme in the presence of betaine and trehalose were studied by tryptophan flurescence spectroscopy. Equilibrium stability parameters of lysozyme unfolding were obtained using the linear extrapolation model. It was found that the free energy of unfolding was enhanced by increasing the concentrations of both the osmolytes, resulting in the thermodynamic stabilization of lysozyme.
Second, betaine and trehalose were used as folding aids to assist the renaturation of denatured-reduced lysozyme. It was observed that the refolding yield was improved with the increase in betaine concentration up to 2 mol·L-1, while it appeared to present an optimal trehalose concentration at about 0.2 mol·L-1. Then, the kinetic behavior of the refolding process was studied at different guanidinium chloride concentrations, and the dynamic process was expressed by the competitive model of first-order folding reaction and third-order aggregation. In the presence of betaine, the refolding rate was accelerated, leading to the increase of the refolding yield. The unfolded proteins could be stabilized by trehalose, and the aggregation of lysozyme was suppressed at low trehalose concentrations (<0.2 mol·L-1), thus facilitating the refolding. However, lysozyme renaturation was inhibited at high trehalose concentrations (>0.2 mol·L-1), showing low folding rates revealed by the kinetic model.
Third, to reveal the effects of the osmolytes on the intermolecular interactions during protein refolding, self-interaction chromatography was employed to study the effect of betaine, trehalose and arginine on the second virial coefficient, BB22, of lysozyme in solutions containing guanidinium chloride or NaCl at different pH values. At all guanidinium chloride concentrations (0~6 mol·L- 1), B22 increased by raising betaine and trehalose concentrations. With increasing arginine concentrations, B22 decreased at low guanidinium chloride concentrations, but increased at high guanidinium chloride concentrations. The results indicated the different effects of the osmolytes from that of arginine in the intermolecular interactions.
Finally, recombinant E. coli malate dehydrogenase (eMDH) was expressed as inclusion bodies (IBs), and the eMDH IBs was used as a real oligomeric protein to study protein refolding from IBs. It was observed that the specific activity of eMDH was enhanced by increasing betaine concentration, while there was an optimal trehalose concentration (0.5 mol·L-1) at which the protein was favorably renatured. Furthermore, the renaturation could be improved even at high guanidinium chloride concentrations (up to 0.4 mol·L-1) in the presence of betaine or trehalose.
引文
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