【作者】 王超展；

【导师】 耿信笃；

【作者基本信息】 西北大学 ， 分析化学， 2004， 博士

【摘要】 基因重组蛋白的复性是生物工程下游技术中的一个关键问题,因长期未能得到很好的解决,从而在很大程度上制约着生物工程产品的产业化。用液相色谱（LC）进行变性蛋白复性是近年来迅速发展起来的一种新的方法,它能够显著地提高蛋白复性效率,并同时使目标蛋白得到纯化,目前已成为国际上的研究热点。本论文应用LC对两种重组蛋白,重组牛朊病毒蛋白片断[rbPrP（104-242）]和重组人粒细胞集落刺激因子（rhG-CSF）进行了复性并同时纯化,同时还对rhG-CSF的复性并同时纯化工艺进行了放大。 论文包括以下八个部分: 1.文献综述:简要地介绍了蛋白质复性的意义,对近年来发展起来的蛋白质复性新方法进行了系统的综述。也对朊病毒和rhG-CSF的复性及分离纯化研究进展进行了评述。引用文献200篇。 2.用高效疏水色谱（HPHIC）对大肠杆菌（E.coli）表达的重组牛朊病毒蛋白片断rbPrP（104-242）进行了纯化,研究了pH值、固定相和盐种类、梯度模式等几种因素对纯化结果的影响。经一步HPHIC,在40min内可以得到纯度为96%,质量回收率为87%的rbPrP（104-242）,该方法简单高效。圆二色性光谱结果表明所得rbPrP（104-242）的二级结构正确。因此,rbPrP（104-242）可以在HPHIC过程中得到纯化并同时复性。 3.成功地用普通SEC对E.coli表达的rhG-CSF进行了复性并进行了部分纯化,研究了流动相中脲浓度、流动相pH、流速、谷胱甘肽浓度和氧化还原比例,以及甘油浓度等因素对rhG-CSF的SEC复性的影响。在优化的条件下,所得rhG-CSF的比活为1.2×10⁸IU·mg^-1,其质量回收率为30.1%,所用时间为25min。采用脲梯度SEC对rhG-CSF进行了复性,研究了脲梯度的长度和最终的脲浓度对rhG-CSF复性的影响。与普通SEC相比,在流动相中有脲存在并用脲梯度是一种更有效的SEC复性rhG-CSF的方法,能够更好地抑制复性过程中聚集体的产,所得rhG-CSF的比活为1.2×10⁸IU·mg^-1,纯度为82.1%质量回收率为52.8%,所用时间为30min。更多还原

【Abstract】 Refolding of recombinant therapeutic proteins remain a key puzzle in the downstream processing of biotechnology, which restricts the industrialization of bioengineering products at a great extent. Refolding of denatured proteins by liquid chromatography (LC) is a newly developed technique. It can improve the refolding yield and simultaneously purify the target proteins, so it has been paid much attention in recent years. In this dissertation, LC was applied to the refolding of two recombinant proteins, recombinant bovine normal prion protein fragment containing residues 104-242 [rbPrP(104-242)] and recombinant human granulocyte colony-stimulating factor (rhG-CSF). In addition, the process for rhG-CSF was enlarged to preparative scale. The dissertation includes eight sections:1. Review: Significance of protein refolding is introduced briefly, and a comprehensive review on recently developed techniques of protein refolding is provided. In addition, recent development of the refolding and purification of prion protein and rhG-CSF was also reviewed. It contains 200 references.2. Recombinant bovine normal prion protein containing residues 104-242, PrP(104-242) expressed in E.coli was purified with simultancously refolded by high performance hydrophobic interaction chromatography (HPHIC). Several factors, pH value, types of stationery phase and salt, and gradient mode, influencing the purification results were investigated. PrP(104-242) with a purity of 96% and a recovery of 87% was yielded by a single HPHIC step of 40 min. The presented method is simple and high efficiency. Circular dichroism spectroscopy shows that the secondary structure of the purified rbPrP(104-242) is correct. Therefore, rbPrP(104-242) was purified with simultaneous renaturation in the HPHIC process.3. rhG-CSF expressed in E.coli was successfully refolded with simultaneouslypartial purification of by size exclusion chromatography (SEC). Several factors, such as the concentration of urea in the mobile phase, pH value, flow rate, concentration of glutathione and ratio of GSH to GSSG, concentration of glycerol, effecting the target protein refolding were investigated in details. With the optimal conditions, the obtained rhG-CSF has a specific activity of 1.2xlO8 IU-mg’1, purity of 82.7%, mass recovery of 30.1% and it takes only 25 min to complete the refolding of rhG-CSF. To increase the mass recovery of the rhG-CSF. urea gradient SEC was also used to refold of rhG-CSF. The effects of the length and final urea concentration of urea gradient on the refolding of rhG-CSF were investigated. Compared to the SEC at a constant urea urea concentration, SEC with a urea gradient was more efficient, in terms of specific bioactivity and mass recovery, because it could suppress aggregates more efficiently, resulting in the specific activity, purity and mass recovery of the refolded rhG-CSF to be 1.2x10 IU-mg’’, 82.1%, and 52.8%, respectively. It takes 30 min to complete the refolding of rhG-CSF.4. rhG-CSF was successfully refolded with simultaneously purified by strong anion exchange chromatography (SAX). Several factors, including urea concentration, pH value, concentration and ratio of reduced/oxidized glutathione (GSH/GSSG) in the mobile phase, as well as the flow rate of the mobile phase, effecting the refolding yield of the denatured/reduced rhG-CSF were investigated in details. With the optimal conditions, it takes only 30 min for rhG-CSF to completely accomplish its refolding and the obtained rhG-CSF has a specific activity of 3.0x108 IU-mg"1, purity of 96.4%, and mass recovery of 49.3%, respectively, while the specific activity and mass recovery of the rhG-CSF obtained by dilution method taking 9 h, the obtained rhG-CSF are 0.93* 108 IU-mg’1 and 32.7%, respectively. Compared to the dilution refolding method, the SAX method has higher refolding efficiency for rhG-CSF, and save many steps, such as dilution, removal of precipitation by centrifugation, concentration of sample solution andsample loading,.5. Weak anion exchange (WAX) chromatograpliic cake was used to the refolding of rhG-CSF with simultaneous purification, the presented method can work well in case of some precipitates forming during sample injecting. Several factors, including urea concentration in the mobile phase, types of salts and buffers, pH, flow rate, redox ratio, influencing the refolding of rhG-CSF were investigated. With the optimal conditions, the obtained rhG-CSF in 25 min has a specific activity of 1.9*108 IU-mg"1, purity of 95.8%, and mass recovery of 9.4%, respectively.6. Immobilized metal ion affinity chromatography (IMAC) was also used to refold rhG-CSF. Several factors, urea concentration in the mobile phase, pH, type of buffer and concentration of glycerol, effecting the refolding were investigated. With the optimal conditions, the obtained rhG-CSF by IMAC in 40 min has a specific activity of 2.3* 108 IU-mg"1, purity of 98.2%, and mass recovery of 36.4%, respectively. It is the first time to use IMAC to refold a non-fusion protein.7. HPHIC was applied to the refolding of rhG-CSF solubilized by 8.0 mol-L’1 urea. Several factors, including types of salts and buffers, concentration of ammonium sulfate, pH, flow rate and stationary phase, effecting the refolding of rhG-CSF were investigated. Under the selected optimal conditions, rhG-CSF was refolded with simultaneous purification with one HPHIC step in 28 min, its specific activity is 2.1 X 108 IUmg"1, purity is 96.8%, and mass recovery of 42.6%, respectively. The process has several attractive advantages, such as low cost, short operational period, easy scale up, and thus has great potential for large scale production of rhG-CSF.8. The process for rhG-CSF refolding by HPHIC was enlarged with a HPHIC chromatographic cake (10 x 200 mm I.D.). Several factors, flow rate, initial concentration of ammonium sulfate, gradient length and sample method, effecting the refolding results were investigated. 200 mL 8.0 mol-L"1 denatured rhG-CSF solution,with a total protein about 1.6 g, can be loaded onto the. HPHIC chromatographic cake (10X200 mm I. D). Under these conditions, the obtained rhG-CSF has specific activity of 2.3><108 IU-mg"1, purity more than 95.4% and mass recovery of 36.9%, respectively. It only takes 140 min to complete the refolding of rhG-CSF in such large scale.In summary, the results from this dissertation indicates that very good results were obtained for rbPrP( 104-242) using HPHIC. For the protein containing two disulfide bonds, rhG-CSF, several kinds of refolding liquid chromatography can all be applied to its refolding with simultaneous purification. The refolding yields of rhG-CSF obtained by SAX, WAX, IMAC and HPHIC, respectively, are higher than those obtained by dilution or SEC, and those obtained by SAX is the best among these LC methods. But because its matrix is not rigid, so it can not be used at high flow rate, resulting in relatively long time for industrial process. Although the mass recovery of rhG-CSF obtained by HPHIC is a little lower than that by urea gradient SEC and comparable to that by SAX, but the HPHIC chromatographic cake method is easy to scale up for industrial process. In addition, either protein precipitates form during sample injecting, or during protein refolding by LC due to the precipitates accumulate gradually, resulting the great increase of the back pressure of LC column, HPHIC chromatographic method can still work well in the two circumstances. The protein precipitates can be recovered by re-dissolving periodically and refolded again. It is pointed out that the contribution of stationary phase to protein refolding is very important by LC.更多还原