多聚化合物在提高蛋白质体外正确折叠及稳定性方面的作用及相关机制研究
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摘要
基因工程的迅猛发展使大规模生产基因重组蛋白质成为可能,但在蛋白药物工程化和产业化发展过程中,面临着两个关键的“瓶颈”问题。一是通过基因工程生产的重组蛋白往往以包涵体形式存在,必须经过复性才能获得最终的目的产物,纵观国内外在蛋白复性方面的研究,目前仍然以经验方法为多,产业化推广价值不大。近几年发展起来的折叠助剂促蛋白复性技术对实现蛋白高效复性创造了新的希望,但绝大多数折叠助剂与复性蛋白的结合是不可逆的,这又给后续蛋白分离纯化带来了诸多困难。二是正确折叠的功能蛋白,在实际应用过程中,常常存在稳定性差、体内半衰期短的缺陷,使得蛋白药物保存困难、临床用药频率高、治病成本高,严重制约了蛋白类药物的产业化及临床应用。
围绕上述“瓶颈”问题,本文开展了两大方面的研究。首先,研究一种新型折叠助剂即智能型多聚化合物Eudragit S-100是否可促进蛋白的复性,并通过与现有方法比较,得出其作用优势,再在此基础上对其作用机制开展深入的研究,具体包括：
1)以溶菌酶(lysozyme)为模型蛋白,从复性蛋白活性、含量以及结构等方面研究了Eudragit S-100对溶菌酶复性促进作用。活性检测结果表明,在Eudragit S-100浓度为0.4%(w/v)时,1mg/mL浓度溶菌酶的复性回收率达到了81%,而未加EudragitS-100的对照组仅为49.7%,荧光光谱和圆二色谱分析也表明,在Eudragit S-100作用下,复性溶菌酶的结构与天然态一致。
2)根据Eudragit S-100的理化特性,采用沉淀反应和离子交换实验证明了EudragitS-100对溶菌酶的促复性作用是基于两者发生了离子结合反应,进而Eudragit S-100遮蔽了溶菌酶折叠中间态的疏水基团、抑制了蛋白间疏水基团相互作用引起的聚集效应。而上述离子反应是可逆的,通过调节pH值和离子交换层析即可实现蛋白与EudragitS-100的高效分离,这无疑显示出与现有其它复性方法的优势。
3)通过对作用机制的研究,本文还得出了一个有意义的推测,即Eudragit S-100仅对碱性蛋白有促复性作用。为了验证这一推测的正确性,选择了成纤维细胞生长因子(FGF)两个典型的家族成员即人酸性FGF(haFGF)和人碱性FGF(hbFGF)为模型蛋白。研究结果表明,Eudragit S-100仅对hbFGF有促复性作用,而对haFGF基无影响,这一结果很好地佐证了上述推测的正确性。此部分研究的最后又将Eudragit S-100应用到另外两种碱性蛋白,即角化细胞生长因子KGF-2和转化生长因子TGF-β1中,也得到了理想的结果。
本文的第二个大部分主要集中于多聚化合物20kDPEG-异硫氰酸(PIT-PEG20K)对提高蛋白稳定性的研究,具体包括：
1)选择角化细胞生长因子KGF-2为模型蛋白,采用上述新型修饰制剂对KGF-2进行定位修饰。通过三因素三水平正交实验,考查了反应物摩尔比、温度和pH值等多因素对PIT-PEG20K修饰KGF-2的影响,最终确定出最佳的修饰条件为：PIT-PEG20K对KGF-2摩尔配比为9：1、反应温度为25℃、pH值为6.0；在该修饰条件下,KGF-2的修饰产率达到31.63%。通过肝素亲和层析分离方法获得了纯度超过99%的修饰蛋白PEG-KGF-2。
2)对修饰的生物学性能进行了系统的研究,结果表明,修饰蛋白相比未修饰蛋白生物学活性保留了60%,热稳定性和结构稳定性均显著提高,蛋白空间结构未发生显著变化,体内半衰期由修饰前的2.6min提高到15.5min；另外,体内免疫原性也显著降低。
总之,通过上述两大部分的研究,为碱性蛋白的高效复性寻求到一种崭新的方法；另外,采用PEG定位修饰的方法,成功获得了稳定性显著提高、体内半衰期显著延长的功能蛋白KGF-2。从而为解决蛋白工程化过程中面临的两大“瓶颈”问题提供了好的思路和手段。
The rapid development of genetic engineering makes large-scale production of recombinant protein as possible, but during the industrial drug development, two critical "bottleneck" problems have to be solved. First, the over-expression of heterologous protein in microorganisms such as Escherichia coli often results in the formation of intracellular insoluble protein, also known as inclusion bodies. These inclusion bodies must be refolded in vitro to gain solubility and bioactivity. However, the experiential methods are still prevalent in the research of protein refolding at home and abroad, which have little value in promotion of industrialization. The refolding additives have been reported to suppress aggregation by preventing the association of refolding intermediates or unfolded species through hydrophobic or ionic interactions. But the interaction of proteins with the vast majority of refolding additives is irreversible, which makes follow-up protein separation and purification have many difficulties. Second, in the practical application of the process, the correct folding of functional proteins has the defect such as poor stability and short half-life in vivo. This makes protein drugs difficulty to conserve, high frequency use and high cost of medical treatment, which restrict severely the protein drugs industrialization and clinical application.
To address the above-mentioned "bottleneck" problem, this paper carried out two studies: First, a new type of refolded additive was studied on an intelligent poly-compound Eudragit S-100:whether it can promote protein refolding, obtain its role of advantages compared with existing methods. On this basis, the mechanisms of Eudragit-assisted protein refolding were investigated, specifically including:
1. It was studied whether Eudragit S-100 can promote protein refolding from the activity, content and structure of refolded protein, with lysozyme as a model protein. This study showed that (ⅰ) the addition of Eudragit S-100 in the refolding buffer significantly increased lysozyme refolding yield to 81%, when the concentration of it was up to 0.4%(w/v) and dilution refolding was conducted at 1 mg/mL lysozyme; (ⅱ) Eudragit-lysozyme interaction did not compromise the refolded protein conformation, as confirmed by fluorescence and circular dichroism spectroscopy.
2. According to the physical and chemical properties of Eudragit S-100. precipitation and ion-exchange experiments showed the evidence of an electrostatic interaction between oppositely charged lysozyme and the Eudragit S-100 polymer during refolding. These ionic complexes of Eudragit S-100 and lysozyme appeared to shield expose hydrophobic residues of the lysozyme refolding intermediates, thus minimizing hydrophobic-driven aggregation of the molecules. Importantly, due to the electrostatic interaction is reversible; the polymer can be readily dissociated from the protein by ion exchange chromatography.
3. Through the molecular mechanism of Eudragit S-100 promoting protein refolding, we found an interesting speculation that Eudragit S-100 can only promote the refolding of basic proteins. In order to verify the correctness of this speculation, we selected two ionic forms of human fibroblast growth factor:(ⅰ) human basic fibroblast growth factor (hbFGF) and (ii) human acidic fibroblast growth factor (haFGF) as model proteins. The results showed that Eudragit S-100 only enhanced the refolding yield of hbFGF, but little effect on haFGF. Finally, we successfully applied this strategy to refold another two basic proteins KGF-2 (keratinocyte growth factor-2) and TGF-β1 (transforming growth factor-(31).
The second part focused on the study of PEG-phenyl-isothiocyanate enhancing protein stability studies, specifically including:
1. In present study, we modified keratinocyte growth factor-2(KGF-2) by PEGylation at the N-terminal residue using the above-mentioned PEGylation reagent. The effects of reactants molar ratio, temperature and pH on the PEGylation were examined through the three-factor three-level orthogonal experiments. The optimized modification conditions ultimately determined:the molar ratio of PIT-PEG20K to KGF-2 was 9:1, the reaction temperature was 25℃, pH value was 6.0. Under the modified conditions, KGF-2 production rate of the modified was 31.63%. Under this condition, the yield of PEGylated rhKGF-2 reached a significant amount of 37.8%. PEGylated KGF-2 was then purified by a Heparin Sepharose TM CL-6B affinity chromatography and its purity was over 99%.
2. The biological properties of PEGylated KGF-2 were systematically studied. The results showed that the PEGylated rhKGF-2 retained about 60% of mitogenic activity compared with the non-modified rhKGF-2. Its relative thermal stability at normal physiological temperature and structural stability were significantly enhanced. Spatial structure of protein had no significant change. The half-life time in vivo prolonged from 2.6 min to 15.5 min. Moreover, the immunogenicity of PEGylated rhKGF-2 in mice decreased significantly as compared with non-modified rhKGF-2.
In conclusion, we found an efficient refolding method by the above-mentioned studies for basic proteins. Furthermore, we also successfully made a more stable KGF-2 by PEG-modified method. These studies provide some good ideas and tools to solve two critical "bottleneck" problems of protein engineering.
围绕上述“瓶颈”问题,本文开展了两大方面的研究。首先,研究一种新型折叠助剂即智能型多聚化合物Eudragit S-100是否可促进蛋白的复性,并通过与现有方法比较,得出其作用优势,再在此基础上对其作用机制开展深入的研究,具体包括：
1)以溶菌酶(lysozyme)为模型蛋白,从复性蛋白活性、含量以及结构等方面研究了Eudragit S-100对溶菌酶复性促进作用。活性检测结果表明,在Eudragit S-100浓度为0.4%(w/v)时,1mg/mL浓度溶菌酶的复性回收率达到了81%,而未加EudragitS-100的对照组仅为49.7%,荧光光谱和圆二色谱分析也表明,在Eudragit S-100作用下,复性溶菌酶的结构与天然态一致。
2)根据Eudragit S-100的理化特性,采用沉淀反应和离子交换实验证明了EudragitS-100对溶菌酶的促复性作用是基于两者发生了离子结合反应,进而Eudragit S-100遮蔽了溶菌酶折叠中间态的疏水基团、抑制了蛋白间疏水基团相互作用引起的聚集效应。而上述离子反应是可逆的,通过调节pH值和离子交换层析即可实现蛋白与EudragitS-100的高效分离,这无疑显示出与现有其它复性方法的优势。
3)通过对作用机制的研究,本文还得出了一个有意义的推测,即Eudragit S-100仅对碱性蛋白有促复性作用。为了验证这一推测的正确性,选择了成纤维细胞生长因子(FGF)两个典型的家族成员即人酸性FGF(haFGF)和人碱性FGF(hbFGF)为模型蛋白。研究结果表明,Eudragit S-100仅对hbFGF有促复性作用,而对haFGF基无影响,这一结果很好地佐证了上述推测的正确性。此部分研究的最后又将Eudragit S-100应用到另外两种碱性蛋白,即角化细胞生长因子KGF-2和转化生长因子TGF-β1中,也得到了理想的结果。
本文的第二个大部分主要集中于多聚化合物20kDPEG-异硫氰酸(PIT-PEG20K)对提高蛋白稳定性的研究,具体包括：
1)选择角化细胞生长因子KGF-2为模型蛋白,采用上述新型修饰制剂对KGF-2进行定位修饰。通过三因素三水平正交实验,考查了反应物摩尔比、温度和pH值等多因素对PIT-PEG20K修饰KGF-2的影响,最终确定出最佳的修饰条件为：PIT-PEG20K对KGF-2摩尔配比为9：1、反应温度为25℃、pH值为6.0；在该修饰条件下,KGF-2的修饰产率达到31.63%。通过肝素亲和层析分离方法获得了纯度超过99%的修饰蛋白PEG-KGF-2。
2)对修饰的生物学性能进行了系统的研究,结果表明,修饰蛋白相比未修饰蛋白生物学活性保留了60%,热稳定性和结构稳定性均显著提高,蛋白空间结构未发生显著变化,体内半衰期由修饰前的2.6min提高到15.5min；另外,体内免疫原性也显著降低。
总之,通过上述两大部分的研究,为碱性蛋白的高效复性寻求到一种崭新的方法；另外,采用PEG定位修饰的方法,成功获得了稳定性显著提高、体内半衰期显著延长的功能蛋白KGF-2。从而为解决蛋白工程化过程中面临的两大“瓶颈”问题提供了好的思路和手段。
The rapid development of genetic engineering makes large-scale production of recombinant protein as possible, but during the industrial drug development, two critical "bottleneck" problems have to be solved. First, the over-expression of heterologous protein in microorganisms such as Escherichia coli often results in the formation of intracellular insoluble protein, also known as inclusion bodies. These inclusion bodies must be refolded in vitro to gain solubility and bioactivity. However, the experiential methods are still prevalent in the research of protein refolding at home and abroad, which have little value in promotion of industrialization. The refolding additives have been reported to suppress aggregation by preventing the association of refolding intermediates or unfolded species through hydrophobic or ionic interactions. But the interaction of proteins with the vast majority of refolding additives is irreversible, which makes follow-up protein separation and purification have many difficulties. Second, in the practical application of the process, the correct folding of functional proteins has the defect such as poor stability and short half-life in vivo. This makes protein drugs difficulty to conserve, high frequency use and high cost of medical treatment, which restrict severely the protein drugs industrialization and clinical application.
To address the above-mentioned "bottleneck" problem, this paper carried out two studies: First, a new type of refolded additive was studied on an intelligent poly-compound Eudragit S-100:whether it can promote protein refolding, obtain its role of advantages compared with existing methods. On this basis, the mechanisms of Eudragit-assisted protein refolding were investigated, specifically including:
1. It was studied whether Eudragit S-100 can promote protein refolding from the activity, content and structure of refolded protein, with lysozyme as a model protein. This study showed that (ⅰ) the addition of Eudragit S-100 in the refolding buffer significantly increased lysozyme refolding yield to 81%, when the concentration of it was up to 0.4%(w/v) and dilution refolding was conducted at 1 mg/mL lysozyme; (ⅱ) Eudragit-lysozyme interaction did not compromise the refolded protein conformation, as confirmed by fluorescence and circular dichroism spectroscopy.
2. According to the physical and chemical properties of Eudragit S-100. precipitation and ion-exchange experiments showed the evidence of an electrostatic interaction between oppositely charged lysozyme and the Eudragit S-100 polymer during refolding. These ionic complexes of Eudragit S-100 and lysozyme appeared to shield expose hydrophobic residues of the lysozyme refolding intermediates, thus minimizing hydrophobic-driven aggregation of the molecules. Importantly, due to the electrostatic interaction is reversible; the polymer can be readily dissociated from the protein by ion exchange chromatography.
3. Through the molecular mechanism of Eudragit S-100 promoting protein refolding, we found an interesting speculation that Eudragit S-100 can only promote the refolding of basic proteins. In order to verify the correctness of this speculation, we selected two ionic forms of human fibroblast growth factor:(ⅰ) human basic fibroblast growth factor (hbFGF) and (ii) human acidic fibroblast growth factor (haFGF) as model proteins. The results showed that Eudragit S-100 only enhanced the refolding yield of hbFGF, but little effect on haFGF. Finally, we successfully applied this strategy to refold another two basic proteins KGF-2 (keratinocyte growth factor-2) and TGF-β1 (transforming growth factor-(31).
The second part focused on the study of PEG-phenyl-isothiocyanate enhancing protein stability studies, specifically including:
1. In present study, we modified keratinocyte growth factor-2(KGF-2) by PEGylation at the N-terminal residue using the above-mentioned PEGylation reagent. The effects of reactants molar ratio, temperature and pH on the PEGylation were examined through the three-factor three-level orthogonal experiments. The optimized modification conditions ultimately determined:the molar ratio of PIT-PEG20K to KGF-2 was 9:1, the reaction temperature was 25℃, pH value was 6.0. Under the modified conditions, KGF-2 production rate of the modified was 31.63%. Under this condition, the yield of PEGylated rhKGF-2 reached a significant amount of 37.8%. PEGylated KGF-2 was then purified by a Heparin Sepharose TM CL-6B affinity chromatography and its purity was over 99%.
2. The biological properties of PEGylated KGF-2 were systematically studied. The results showed that the PEGylated rhKGF-2 retained about 60% of mitogenic activity compared with the non-modified rhKGF-2. Its relative thermal stability at normal physiological temperature and structural stability were significantly enhanced. Spatial structure of protein had no significant change. The half-life time in vivo prolonged from 2.6 min to 15.5 min. Moreover, the immunogenicity of PEGylated rhKGF-2 in mice decreased significantly as compared with non-modified rhKGF-2.
In conclusion, we found an efficient refolding method by the above-mentioned studies for basic proteins. Furthermore, we also successfully made a more stable KGF-2 by PEG-modified method. These studies provide some good ideas and tools to solve two critical "bottleneck" problems of protein engineering.
引文
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