蛋白折叠液相色谱法复性与同时纯化重组人Flt3配体的研究
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摘要
近年来,蛋白折叠液相色谱(Protein folding liquid chromatography, PFLC)法越来越多地被应用于包涵体蛋白复性与同时纯化中,PFLC的特点是能在色谱过程将目标蛋白与其它组分分开的同时,还能在色谱柱上进行变性蛋白折叠。本论文①对在大肠杆菌(E.coli)中表达的重组人酪氨酸激酶配体(Recombined human Flt3 ligand, rhFL)进行了摇瓶中菌种培养条件的优化,获得了高表达的rhFL包涵体蛋白；②经超声破碎、离心并多次洗涤沉淀获得纯化的包涵体,并将其溶解于8 mol／L脲中得到变性蛋白的抽提液；③利用高效疏水相互作用色谱(HPHIC)法,包括一个分析型HPHIC柱、半制备型HPHIC饼和新型单柱二维液相色谱(A novel two-dimensional liquid chromatography using a single column,2DLC-1C)柱进行rhFL包涵体复性与同时纯化的保留特征和复性规律的研究,为大规模制备rhFL蛋白和将其用于临床研究奠定基础。
本论文共有四个部分：
1.文献综述：对近年来不同机理的PFLC法在蛋白质折叠与同时纯化中的进展和应用,以及FL的结构、生物学功能及临床应用的重要性进行了综述。
2.摇瓶中rhFL菌种培养条件的优化：研究了摇瓶中培养基、培养条件(温度、诱导时间、pH、接种量)对rhFL表达的影响。实验结果表明,在培养温度为35℃、选用初始pH值为6.6的SOB培养基、甘油做为碳源、诱导4 h、接种量为4%的条件下,rhFL蛋白的表达量可占菌体总蛋白的47.4%。
3. HPHIC法对E.coli表达的rhFL包涵体复性与同时纯化：分别选用不同规格的色谱柱(分析型色谱柱、半制备型色谱饼),通过色谱流动相(小分子添加剂、流速)和进样量对rhFL复性与纯化过程中质量回收率的影响,筛选出rhFL包涵体蛋白在HPHIC柱上复性与纯化的最佳条件和保留规律,初步用荧光光谱法考察了rhFL在色谱柱上复性前后荧光强度变化,并用HPHIC饼对rhFL进行规模化制备。结果分别在固定相配基为苯基的分析型色谱柱、半制备色谱饼上复性与同时纯化rhFL包涵体的质量回收率可达45.9%、67.0%,纯度分别为93.5%、92.1%。对G-CSF依赖株NFS-60的细胞增殖作用的活性结果表明,当与G-CSF协同作用时,平均比活为7.42×108IU／mg。
4.新型单柱二维色谱柱对rhFL包涵体复性与同时纯化：新型2DLC-1C柱是在一根色谱柱上兼具有了HIC和IEC两种保留模式,已被用于活性蛋白的快速分离。本工作选用了2DLC-1C柱的HIC模式与和以苯基为配基的HPHIC柱,比较两种色谱柱对rhFL复性与同时纯化过程中流动相的洗脱的盐浓度变化对目标蛋白质量回收率的影响。实验结果显示,在以苯基为配基的色谱柱上,当流动相(NH4)2SO4盐浓度为3.0 mol／L时,rhFL的质量回收率可达到48.7%。而新型的2DLC一1C柱,当流动相为2.0 mol／L(NH4)2SO4时,即可获得的质量回收率为48.5%。从而证实新型填料的2DLC-1C柱在较低盐浓度下可达到较高的质量回收率。此结果也提示这种新型的双保留模式色谱柱,有可能降低规模化制备rhFL蛋白的成本。
Protein folding liquid chromatography (PFLC) is increasingly being applied to the refolding and simultaneous purification of inclusion body proteins in recent years. The characteristics of PFLC are that it can not only separate the target protein from other constituents, but also make denatured protein folding in the column. In this dissertation,①the shaking flask culture conditions of recombinant human Flt3 ligand (rhFL) expressed in E.coli were studied, and high-level expressed rhFL inclusion bodies were obtained.②The cells were disrupted by ultrasonication, purified inclusion bodies were colleceted after centrifugation and repeated washing and were dissolved in 8 mol/L urea, obtaining the denatured protein extracts.③Retention feature and renaturation rules of rhFL in the process of refolding and simultaneous purification were investigated by using high performance hydrophobic interaction chromatography (HPHIC) method, in which an analytical HPHIC column, a semi-preparative HPHIC cake and a novel two-dimensional liquid chromatography by a single column were separately employed, this provided a foundation for large scale preparation of rhFL protein and its clinical research applications.
The dissertation consists of four sections:
1. Literature review:The recent advance and applications of PFLC methods with different mechanisms in the protein refolding and simultaneous purification were reviewed, and the FL structure, biological functions, its importance in clinical applications were also reviewe.
2. Optimization of conditions for expressing rhFL in the shaking flask:-Influence of culture media in the shaking flask, culture conditions (temperature, inducing time, pH, inoculation amount) on the expression of rhFL were investigated. The experimental results showed that when the culture temperature was 35℃, initial pH of SOB culture medium was 6.6, glycerol was selected as a carbon source, inducing time was 4 h, inoculation amount was 4%, the expression amount of rhFL protein could be up to 47.4% of the total bacterial protein.
3. Refolding and simultaneous purification of rhFL from inclusion bodies expressed in E.coli by HPHIC:Chromatographic columns with different dimensions (analytical chromatographic column, semi-preparative chromatographic cake) were separately used, effects of chromatographic mobile phase (small molecular additives, flow rate) and loading volume on the mass recovery of rhFL during the refolding and purification were investigated, the optimum conditions and retention rules of the renaturation and purification of rhFL on HPHIC column were obtained, fluorescence intensity variations of rhFL inclusion bodies before and after the refolding in the chromatographic column were preliminarily investigated by fluorescence spectrometry, and rhFL was prepared in the large scale by using HPHIC cake. The results showed that the mass recovery of rhFL could reach to 45.9% and 67.0% respectively after refolded and purified by using the analytical chromatographic column and the semi-preparative chromatographic cake, both being packed with stationary phase with phenyl ligands, the purity were 93.5% and 92.1% respectively. The activity results of cell proliferation on G-CSF dependent cell line NFS-60 showed that the average specific bioactivity of rhFL was 7.42×108 IU/mg when synergized with G-CSF.
4. Refolding and simultaneous purification of rhFL inclusion bodies by a novel two-dimensional liquid chromatography:2DLC-1C comumn, which was a novel single column with both HIC and IEC retention models, has been employed for fast separation of active proteins. In this work, a 2DLC-1C column was used in HIC mode to investigate effect of salt concentration in the mobile phase on the renaturation and simultaneous purification of rhFL, and the results were compared with a chromatographic column with phenyl ligands. On the chromatographic column with phenyl ligands, when the (NH4)2SO4 concentration in the mobile phase was 3.0 mol/L, the mass recovery of rhFL was 48.7%, while the mass recovery was 48.5% on the 2DLC-1C column when the (NH4)2SO4 concentration in the mobile phase was 2.0 mol/L, which shows that a relatively high mass recovery can be obtained on the 2DLC-1C column at lower salt concentration. This makes it possible to reduce cost in preparation of rhFL.
本论文共有四个部分：
1.文献综述：对近年来不同机理的PFLC法在蛋白质折叠与同时纯化中的进展和应用,以及FL的结构、生物学功能及临床应用的重要性进行了综述。
2.摇瓶中rhFL菌种培养条件的优化：研究了摇瓶中培养基、培养条件(温度、诱导时间、pH、接种量)对rhFL表达的影响。实验结果表明,在培养温度为35℃、选用初始pH值为6.6的SOB培养基、甘油做为碳源、诱导4 h、接种量为4%的条件下,rhFL蛋白的表达量可占菌体总蛋白的47.4%。
3. HPHIC法对E.coli表达的rhFL包涵体复性与同时纯化：分别选用不同规格的色谱柱(分析型色谱柱、半制备型色谱饼),通过色谱流动相(小分子添加剂、流速)和进样量对rhFL复性与纯化过程中质量回收率的影响,筛选出rhFL包涵体蛋白在HPHIC柱上复性与纯化的最佳条件和保留规律,初步用荧光光谱法考察了rhFL在色谱柱上复性前后荧光强度变化,并用HPHIC饼对rhFL进行规模化制备。结果分别在固定相配基为苯基的分析型色谱柱、半制备色谱饼上复性与同时纯化rhFL包涵体的质量回收率可达45.9%、67.0%,纯度分别为93.5%、92.1%。对G-CSF依赖株NFS-60的细胞增殖作用的活性结果表明,当与G-CSF协同作用时,平均比活为7.42×108IU／mg。
4.新型单柱二维色谱柱对rhFL包涵体复性与同时纯化：新型2DLC-1C柱是在一根色谱柱上兼具有了HIC和IEC两种保留模式,已被用于活性蛋白的快速分离。本工作选用了2DLC-1C柱的HIC模式与和以苯基为配基的HPHIC柱,比较两种色谱柱对rhFL复性与同时纯化过程中流动相的洗脱的盐浓度变化对目标蛋白质量回收率的影响。实验结果显示,在以苯基为配基的色谱柱上,当流动相(NH4)2SO4盐浓度为3.0 mol／L时,rhFL的质量回收率可达到48.7%。而新型的2DLC一1C柱,当流动相为2.0 mol／L(NH4)2SO4时,即可获得的质量回收率为48.5%。从而证实新型填料的2DLC-1C柱在较低盐浓度下可达到较高的质量回收率。此结果也提示这种新型的双保留模式色谱柱,有可能降低规模化制备rhFL蛋白的成本。
Protein folding liquid chromatography (PFLC) is increasingly being applied to the refolding and simultaneous purification of inclusion body proteins in recent years. The characteristics of PFLC are that it can not only separate the target protein from other constituents, but also make denatured protein folding in the column. In this dissertation,①the shaking flask culture conditions of recombinant human Flt3 ligand (rhFL) expressed in E.coli were studied, and high-level expressed rhFL inclusion bodies were obtained.②The cells were disrupted by ultrasonication, purified inclusion bodies were colleceted after centrifugation and repeated washing and were dissolved in 8 mol/L urea, obtaining the denatured protein extracts.③Retention feature and renaturation rules of rhFL in the process of refolding and simultaneous purification were investigated by using high performance hydrophobic interaction chromatography (HPHIC) method, in which an analytical HPHIC column, a semi-preparative HPHIC cake and a novel two-dimensional liquid chromatography by a single column were separately employed, this provided a foundation for large scale preparation of rhFL protein and its clinical research applications.
The dissertation consists of four sections:
1. Literature review:The recent advance and applications of PFLC methods with different mechanisms in the protein refolding and simultaneous purification were reviewed, and the FL structure, biological functions, its importance in clinical applications were also reviewe.
2. Optimization of conditions for expressing rhFL in the shaking flask:-Influence of culture media in the shaking flask, culture conditions (temperature, inducing time, pH, inoculation amount) on the expression of rhFL were investigated. The experimental results showed that when the culture temperature was 35℃, initial pH of SOB culture medium was 6.6, glycerol was selected as a carbon source, inducing time was 4 h, inoculation amount was 4%, the expression amount of rhFL protein could be up to 47.4% of the total bacterial protein.
3. Refolding and simultaneous purification of rhFL from inclusion bodies expressed in E.coli by HPHIC:Chromatographic columns with different dimensions (analytical chromatographic column, semi-preparative chromatographic cake) were separately used, effects of chromatographic mobile phase (small molecular additives, flow rate) and loading volume on the mass recovery of rhFL during the refolding and purification were investigated, the optimum conditions and retention rules of the renaturation and purification of rhFL on HPHIC column were obtained, fluorescence intensity variations of rhFL inclusion bodies before and after the refolding in the chromatographic column were preliminarily investigated by fluorescence spectrometry, and rhFL was prepared in the large scale by using HPHIC cake. The results showed that the mass recovery of rhFL could reach to 45.9% and 67.0% respectively after refolded and purified by using the analytical chromatographic column and the semi-preparative chromatographic cake, both being packed with stationary phase with phenyl ligands, the purity were 93.5% and 92.1% respectively. The activity results of cell proliferation on G-CSF dependent cell line NFS-60 showed that the average specific bioactivity of rhFL was 7.42×108 IU/mg when synergized with G-CSF.
4. Refolding and simultaneous purification of rhFL inclusion bodies by a novel two-dimensional liquid chromatography:2DLC-1C comumn, which was a novel single column with both HIC and IEC retention models, has been employed for fast separation of active proteins. In this work, a 2DLC-1C column was used in HIC mode to investigate effect of salt concentration in the mobile phase on the renaturation and simultaneous purification of rhFL, and the results were compared with a chromatographic column with phenyl ligands. On the chromatographic column with phenyl ligands, when the (NH4)2SO4 concentration in the mobile phase was 3.0 mol/L, the mass recovery of rhFL was 48.7%, while the mass recovery was 48.5% on the 2DLC-1C column when the (NH4)2SO4 concentration in the mobile phase was 2.0 mol/L, which shows that a relatively high mass recovery can be obtained on the 2DLC-1C column at lower salt concentration. This makes it possible to reduce cost in preparation of rhFL.
引文
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