以木瓜蛋白酶为模型蛋白的亲和色谱新介质的制备及其吸附机理研究
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摘要
亲和色谱是一种利用生物大分子和固定相表面存在某种特异性亲和力,选择性地分离目标分离物的现代分离纯化技术。随着这些年高效亲和色谱技术的发展与成熟,使其在有机化学、生物化学、分子生物学、生物工程、基因工程等领域的应用受到越来越广泛的关注。然而由于亲和配基价格昂贵、色谱操作压大、处理量小等缺点,使之很难在低成本,高效率的前提下发挥更大的作用,因此寻找具有亲和配基相似活性的小分子和发展新型廉价的载体材料成为亟待解决的问题。目前,通过组合筛选技术筛选亲和配基和改良现有的色谱载体的方法占有主导地位,但是组合筛选技术很大程度上增加了筛选配基的工作量和成本,改良现有载体更是无法从根本上解决操作繁琐,处理量小等缺陷。通过选择价格低廉的染料或金属离子为亲和配基制备新型色谱介质,以及用生物技术淘洗亲和配基的方法可以很好地解决上述两个难题,而且可以在保证不降低目标分离物生物活性的前提下,快速、高效地进行分离纯化操作。在此方面的研究中,选择染料亲和色谱和仿生配基亲和色谱,发展新型载体等课题引起了广大研究学者的关注。
本文以木瓜蛋白酶为模型蛋白,分别通过化学法和生物法制备了两种能够低成本,高效率地分离纯化模型蛋白的亲和色谱新介质,即通过化学法制备了利用膜色谱的低压降,染料配基的低成本的染料亲和膜色谱介质；通过生物法制备了利用噬菌体展示技术的高效率,噬菌体交联法的低损耗的交联噬菌体七肽亲和吸附介质(CAPOP)。分别考察了这两种亲和色谱介质对模型蛋白的吸附性能,利用亲和吸附模型阐述了两种亲和色谱介质对模型蛋白的吸附机理,并最终评价了对模型蛋白分离纯化的有效性。
本文首先以机械强度好的尼龙膜为载体,利用化学法选择1M HCl酸水解法、甲醛活化法、壳聚糖改性法,键合染料后制备出染料Reactive Red120亲和膜色谱介质。通过扫描电镜、紫外光谱、红外光谱和元素分析对染料亲和膜进行定性定量分析,确定染料亲和膜壳聚糖含量为118.5+7.5mg/g,染料含量为163.4+10.3μmol/g,孔径大小均匀(0.5-3.0μm),比表面积大,利于蛋白质分子自由通过,是一种较为理想的分离纯化介质。
以木瓜蛋白酶为模型蛋白,采用静态吸附法和动态脱附法研究染料亲和膜色谱介质的蛋白质吸附性能。静态吸附法考察了配基供给量、缓冲液pH值、缓冲液钠离子强度、吸附温度和吸附时间等对木瓜蛋白酶吸附量的影响。在吸附条件为：染料配基浓度10.0mg/mL、缓冲液pH8.5、钠离子强度为0M、吸附温度为37℃、吸附3h时最大静态吸附量达到37.0mg/g。动态脱附法考察了洗脱液、洗脱液pH、洗脱液钠离子强度和洗脱速度等对木瓜蛋白酶脱附量的影响。在动态脱附条件为NaCl洗脱液pH6.0,钠离子强度为1.0M,洗脱速度为1mL/min地洗脱下,达到最大的动态脱附效果,纯化后的酶活力达到1035.53U/mg,纯化倍数约为20倍。
采用吸附等温模型、吸附动力学模型、吸附热力学模型和计算机模拟实验阐述染料亲和膜对模型蛋白木瓜蛋白酶的吸附机理。以Langmuir吸附模型和Freundlich吸附模型考察染料亲和膜的蛋白质吸附行为。研究发现Freundlich吸附模型可以较好地解释染料亲和膜的吸附行为,证实亲和膜对木瓜蛋白酶的吸附是不均匀立体结构的多分子层吸附模型。吸附动力学模型分别用准一级动力学吸附模型、准二级动力学吸附模型、粒子内扩散吸附模型以及Elovich动力学吸附模型考察染料亲和膜的吸附行为。研究发现准二级动力学吸附模型可以较好解释该吸附行为,证实染料亲和膜表面染料配基的活性位点越多,吸附能力就越强,一旦染料配基的活性位点覆盖率变大,可供共价结合的位点减少,吸附能力下降。吸附热力学研究结果为：焓变值ΔH0、熵变值ΔS0、吉布斯自由能ΔG0分别为18.52kJ/mol,0.077kJ/(mol K)和-4.43kJ/mol(298K)o焓变值ΔH0为18.52kJ/mol表明该吸附过程为吸热反应,温度的增加有利于吸附行为的发生,吸附作用力包括化学作用和物理作用。熵变值ΔS0为0.077kJ/(mol K)表明吸附过程是一个染料配基表面先脱附水分子,再吸附蛋白分子的循环过程,即“溶剂置换作用”,同时证实整个体系的吸附过程是混乱度增加的吸热过程。吉布斯自由能AG为-4.43kJ/mol(298K)不仅证明了吸附行为是自发的,还证实物理作用与化学作用同样存在于该吸附行为中,并发挥主要作用。采用Maestro9.0软件对染料配基和木瓜蛋白酶进行分子对接计算机模拟实验,结果证实吸附行为不仅包含物理吸附(疏水作用),还包括化学偶联作用(氢键)。
染料Reactive Red120亲和膜初步应用于木瓜蛋白酶的分离纯化。通过上样,分离,洗涤,洗脱四个步骤,收集酶活最高的一组组分,冷冻干燥后经比酶活计算和FPLC表征,得到纯化倍数为粗酶溶液22.60倍的木瓜蛋白酶粉,证实所制备的染料Reactive Red120亲和膜能有效的用于木瓜蛋白酶的富集和分离,能满足皮革、饲料等领域对中等纯度的木瓜蛋白酶需求。
论文同时以木瓜蛋白酶为模型,通过噬菌体展示技术筛选的七肽(Ile-Gln-Ser-Pro-His-Phe-Phe)为配基,以戊二醛为交联剂,以交联噬菌体为载体,利用生物法制备出交联噬菌体七肽亲和吸附介质(CAPOP)。通过透射电镜、动态光散射仪、酶联免疫吸附检测对CAPOP进行定性分析,确定七肽配基的ELISAO0492值为1.9,证实对木瓜蛋白酶具有亲和作用,交联噬菌体团聚物的粒径为5-6μm,是单个噬菌体粒径的10倍左右,证实交联成功。该介质具备特异性吸附高,价格低廉,制备简易的优点。
采用静态吸附法、响应面优化法和动态脱附法研究CAPOP的蛋白吸附性能。静态吸附法主要考察了缓冲液pH值、缓冲液钠离子强度、吸附温度和吸附时间等对木瓜蛋白酶吸附量的影响。在吸附条件为缓冲液pH7.0,钠离子强度为0M,吸附温度为25℃,吸附2h时最大静态吸附量达到50mg/g。采用响应面法分析了吸附过程中多种因素共作用对木瓜蛋白酶吸附量的关系。研究发现不同因素的共作用对吸附量有显著影响,得到回归方程并建立优化条件：噬菌体展示配基含量为1011PFU/mL,pH值为6.9,温度为32℃,木瓜蛋白酶粗酶溶液浓度为7.51mg/mL,木瓜蛋白酶的吸附量最高,为55.4229mg/g。动态脱附法考察了洗脱液,洗脱液pH、洗脱液钠离子强度和洗脱速度对木瓜蛋白酶吸附量的影响。在动态脱附条件为pH9.0,钠离子强度为1.0M,洗脱速度为1.0mL/min的NaSCN溶液的洗脱下,达到最大的动态脱附效果,纯化倍数达200倍左右,纯化后的酶活力达到4647.73U/mg,基本与商品酶酶活持平。
采用吸附等温线模型,吸附动力学模型,吸附热力学模型和计算机模拟技术探讨交联噬菌体对木瓜蛋白酶的吸附机理。分别以Langmuir吸附模型和Freundlich吸附模型考察CAPOP的吸附行为。研究发现Freundlich吸附模型可以较好地解释CAPOP的吸附行为,证实CAPOP对木瓜蛋白酶的吸附是不均匀立体结构的多分子层吸附模型,这与交联噬菌体的立体结构相吻合。吸附动力学模型分别以准一级动力学吸附模型、准二级动力学吸附模型和Elovich动力学吸附模型考察CAPOP的吸附行为。研究发现准二级动力学吸附模型可以解释该吸附行为,证实CAPOP表面展示的七肽配基的活性位点越多,吸附能力就越强,一旦七肽配基的活性位点覆盖率变大,可供共价结合的位点减少,吸附能力下降。吸附热力学研究表明：温度在25℃以下,焓变值ΔH0为7.97kJ/mol说明吸附反应为吸热反应,温度地升高有利于吸附行为地发生,作用力包括化学作用和物理作用。ΔS0为0.044kJ/(mol K)表明吸附过程是一个七肽配基表面先脱附水分子,再吸附蛋白分子的循环过程,即“溶剂置换作用”,同时证实整个体系的吸附过程是混乱度增加的吸热过程。吉布斯自由能ΔG0为-5.14kJ/mol(298K)不仅证明了吸附行为是自发的,还证实物理作用与化学作用同样存在于该吸附行为中,并发挥主要作用。在25℃以上,焓变值ΔH0为-12.02kJ/mol,熵变值ΔS0为-0.023kJ/(mol K),熵减焓减,说明吸附体系存在“转变温度”,这可能是由于吸附过程产生了新的化学键。但吉布斯自由能ΔG0为-5.17kJ/mol (298K),仍证明吸附行为是自发的,说明吸附过程存在物理作用与化学作用,并随温度的升高产生了新的化学键。采用Maestro9.0软件对七肽配基和木瓜蛋白酶进行分子对接计算机模拟实验,结果证实吸附行为不仅包含物理吸附(疏水作用),还包括化学偶联作用(氢键作用)。
CAPOP初步应用于木瓜蛋白酶的分离纯化。通过亲和吸附四个步骤,收集酶活最高的组分,冷冻干燥后经比酶活的计算和SDS-PAGE表征,得到纯化倍数为粗酶溶液的240.94倍,是染料亲和膜的近10倍。证实所制备的CAPOP不仅能有效的分离纯化木瓜蛋白酶,并表现为更高的分离效率,具备满足医疗、生物生化工程、抗体工程等领域对高纯度木瓜蛋白酶需求的潜力。
以木瓜蛋白酶为模型蛋白,研究七肽配基对木瓜蛋白酶的抑制行为和抑制机理。通过考察不同浓度的七肽分子对木瓜蛋白酶催化活性的抑制效果,采用Lineweaver-Burk双倒数作图,最终确定当催化体系中七肽分子的浓度达到20mM时,相对抑制率达到了66.41%,得出导致木瓜蛋白酶酶活丧失50%时的七肽分子浓度(IC50)为14.2mM；当七肽分子浓度为20mM时,催化体系的延滞时间为421s,延长了155%。同时证明七肽分子对木瓜蛋白酶酶活的抑制作用是属于可逆性抑制。
综上所述,本论文分别通过化学法和生物法制备两种新型的亲和色谱介质,设计思路具有典型性和拓展性,可推广到任一目标分离物(主要是蛋白质)的特异性配基筛选和规模性纯化,这为新型生物多肽配基的开发、亲和色谱分离过程研究、分离工艺流程选择和设计提供了相关基础数据和理论指导,从而拓宽了亲和色谱技术的应用范围。
Affinity chromatography is a technology of separation and purification, by taking advantage of specific affinity between biological macromolecules and the solid phase surface. With the development of high-performance affinity chromatography, it has been applied in organic chemistry, biochemistry, molecular biology, bioengineering, genetic engineering and other fields. However, due to its expensive affinity ligands, high operating pressure, small handling capacity and other shortcomings, it is difficult to play a much more significant role under the premise of low-cost and high efficiency. To overcome this limit, the developments of the small molecules similar to affinity ligands and new low-cost carrier materials have been widely investigated. More and more scholars are pay attention on screening ligands through a combination method and improving the existing materials, but the former method greatly increases the workload and the cost, and the latter method is unable to overcome the dilemma of complicated operation and small handling capacity. There are good solutions to these problems by adopting dye and metal ion as affinity ligand, or screening for the ligands by biopanning, which could separate the targets proteins rapidly and efficiently and keep the targets high activity. In this regards, the dye and biomimetic affinity chromatography are be concerned by lots of scholars.
Using papain as model protein, two affinity chromatography medium were prepared by chemical and biological methods:dye affinity membrane and cross-linking affinity phage oligopeptide pellet. Adsorption capacity and adsorption mechanism had been studied and the effectiveness of the separation and purification of model protein had been evaluated.
Firstly, using nylon membranes with good mechanical strength as the carrier, covalent coupling of chitosan (CS) to activated nylon membrane was performed after the reaction of the microporous nylon membrane with formaldehyde. The dye Reactive Red120as a ligand was then covalently immobilized on the CS-coated membranes. The dye affinity membranes were characterizied by SEM, UV spectroscopy, FT-IR and elemental analysis. The results showed the chitosan content of dye affinity membranes was determined of118.5±7.5mg/g and Reactive dye Reactive Red120was successfully bond to the modified nylon membrane with the load capacity of163.4±10.3μmol/g. SEM showed the pore sizes of affinity membrane were distributed in the range of about0.5-3.0μm, which facilitated mass transfer. So the dye affinity membrane could be used as an ideal separation and purification media.
The protein adsorption behavior and capacity of the new dye affinity membrane absorbent were studied by static and dynamic method, using papain as a model protein. By testing the different concentrations of ligand, pH, Na ionic strength, temperature and adsorption time, a nice result was achieved:at the circumstances of pH8.5,0M Na ionic strength,37℃, maximum static adsorption of papain was reached to37mg/g after3h. Dynamic testing included elution rate, pH, Na ionic strength and other factors. It showed in the condition of pH6.0,1M ionic strength, elution rate of1mL/min, the papain with the highest activity (1035.53U/mg) would be eluted with20-fold purification.
Adsorption mechanisms of the dye affinity membrane were described by adoption of adsorption isotherm model, adsorption kinetics model, adsorption thennodynamic model and computer simulation study. Adsorption isotherm analysis was identified by Langmuir model and Freundlich model. The linear fit of Freundlich model indicating that the adsorption type is a3-dimensional structure of multi-molecular layer adsorption model. The adsorption kinetics models studies were combined by Pseudo-first-order kinetic model, Pseudo-second-order kinetic model, Intra-particle diffusion model and the Elovich kinetic model. It was found that the Pseudo-second-order kinetic model can better explain the adsorption behavior, indicating that the adsorption capacity was always proportionate to the amounts of active sites on dye affinity membrane surface, once the active sites covered, the adsorption capacity decreased. The values of the standard enthalpy changes, the entropy changes and Gibbs free energy were18.52kJ/mol,0.077kJ/(mol. K),-4.43kJ/mol (298K), respectively. ΔH0value of18.52kJ/mol indicates that the adsorption process is an endothermic reaction, the adsorption capacity increased when temperature elevated and the adsorption force including chemical and physical effects. ΔS0value of0.077kJ/(mol. K) showed the desorption of water molecules occured before adsorption of papain, which named "solvent replacement role", and also confirmed the adsorption process of the system increased disorder. ΔG0value of-4.43kJ/mol (298K) proved that the adsorption behavior was spontaneous, and confirmed that the physical and chemical effects also exist in the adsorption behavior. Computer simulations using the Maestro9.0software also confirmed both the existing of physical (hydrophobic interactions) and chemical effects (hydrogen bonding).
Papain as a model was purified by dye Reactive Red120affinity membrane. Papain was purified22.6-fold in a single step using Red120-CS-membrane, as determined by protein content and enzyme activity assays, meeting papain demands of leather, feed and other areas.
Using7-mer peptides (Ile-Gln-Ser-Pro-His-Phe-Phe) from phage display library as ligand, glutaraldehyde as crosslinking agent and cross-linked phage as matrix, cross-linking affinity phage-oligopeptide pellet (CAPOP) was prepared. Qualitative analysis of enzyme-linked immunosorbent assay confirmed the7-mer peptides ligand have an affinity for papain with OD492value of1.9. Particle size of CAPOP was determined5-6μm by DLS and TEM, indicating the cross-linking successfully. The CAPOP absorbent have the advantages of specific adsorption, low cost and simple preparation.
The adsorption capacity of CAPOP was studied by static adsorption, response surface methodology and dynamic desorption. The static adsorption mainly concerned on the buffer pH, Na ionic strength, the adsorption temperature and adsorption time. The maximum static adsorption capacity reached50mg/g with the optimization of conditions of pH7.0, Na ionic strength0M,25℃and2h. The RSM analysed the relationship between a variety of factors and adsorption capacity as well as establish a model. The maximum papain adsorption capacity reached55.4229mg/g under the conditions of the phage display amount of ligand1011PFU/mL, pH6.9,32℃and papain crude enzyme solution concentration7.51mg/mL. The dynamic desorption concerned about type, pH, Na ionic strength of eluent and the elution speed. A purification of200-fold with its enzyme activity4647.73U/mg was achieved under the dynamic desorption conditions of NaSCN, pH9.0, Na ionic strength1.0M, the elution speed1.0mL/min.
Adsorption mechanisms of CAPOP were studied by adoption of adsorption isotherm model, adsorption kinetics model, adsorption thermodynamic model and computer simulation study. Adsorption isotherm analysis was identified by Langmuir model and Freundlich model. The study showed that the Freundlich model could better explain the adsorption behavior, indicating the adsorption type was a3-dimensional structure of multi-molecular layer adsorption model, which was consistent with the structure of cross-linked phage. The adsorption kinetics models studies were analysed by Pseudo-first-order kinetic model, Pseudo-second-order kinetic model and the Elovich kinetic model. Results showed that the Pseudo-second-order kinetic model could better explain the adsorption behavior, indicating that the adsorption capacity was always proportionate to the amounts of active sites on CAPOP. The values of the standard enthalpy changes, the entropy changes and Gibbs free energy were7.97kJ/mol,0.044kJ/(mol. K),-5.14kJ/mol (298K), respectively. ΔS0value of0.044kJ/(mol. K) showed that the desorption of water molecules occurs before adsorption of papain, which named "solvent replacement role", and also confirmed the adsorption process of the system increased disorder. ΔG0value of-5.14kJ/mol (298K) proved the adsorption behavior was spontaneous, and confirmed that the physical and chemical effects also exist in the adsorption behavior and play a major role. Above25℃, ΔH0value of-12.02kJ/mol, ΔS0value of-0.023kJ/(mol K), indicated that there was a "transition temperature" in adsorption system, which may be due to the new chemical bonds in adsorption process. But ΔG0value of-5.17kJ/mol (298K) still proved the adsorption behavior was spontaneous. Computer simulations using the Maestro9.0software also confirmed both the existing of physical (hydrophobic interactions) and chemical effects (hydrogen bonding).
Papain was purified by CAPOP. Papain was purified240.94-fold in a single step using CAPOP, as determined by protein content and enzyme activity assays. It was confirmed CAPOP was an effective separation and purification absorbents with higher separation efficiency, which have the potential to meet the demands of medical, biological and biochemical engineering, antibody engineering and other fields.
Inhibition mechanism and inhibition behavior of7-mer peptide affinity ligands for papain was studied. Finally it was confirmed the delay time was increasing with the concentration of7-mer peptide increasing, and the7-mer peptide molecule concentration (IC50) was14.2mM by Lineweaver-Burk double reciprocal model. When the catalytic system didn't contain7-mer peptide molecules, the enzyme reaction delay time was165s, whiled the lag time was421s when the concentration of7-mer peptide molecular came to20mM. The results also showed that with the increasing of the7-mer peptide molecules concentration, the catalysis efficiency decreased, indicating that the activity was inhibited by the7-mer peptide molecules, rather than by reducing the amount of enzyme.7-mer peptide molecules on the activity of papain inhibition were identified as irreversible inhibition process.
In summary, we prepared two kinds of affinity chromatography absorbents by chemical and biological methods. It could be expanded any other field of protein separation process including ligand screening, matrix preparation and the design of separation process. Through the study of this thesis, we wish to prodive fundamental data and theory guidence for the application of affinity chromatograph.
本文以木瓜蛋白酶为模型蛋白,分别通过化学法和生物法制备了两种能够低成本,高效率地分离纯化模型蛋白的亲和色谱新介质,即通过化学法制备了利用膜色谱的低压降,染料配基的低成本的染料亲和膜色谱介质；通过生物法制备了利用噬菌体展示技术的高效率,噬菌体交联法的低损耗的交联噬菌体七肽亲和吸附介质(CAPOP)。分别考察了这两种亲和色谱介质对模型蛋白的吸附性能,利用亲和吸附模型阐述了两种亲和色谱介质对模型蛋白的吸附机理,并最终评价了对模型蛋白分离纯化的有效性。
本文首先以机械强度好的尼龙膜为载体,利用化学法选择1M HCl酸水解法、甲醛活化法、壳聚糖改性法,键合染料后制备出染料Reactive Red120亲和膜色谱介质。通过扫描电镜、紫外光谱、红外光谱和元素分析对染料亲和膜进行定性定量分析,确定染料亲和膜壳聚糖含量为118.5+7.5mg/g,染料含量为163.4+10.3μmol/g,孔径大小均匀(0.5-3.0μm),比表面积大,利于蛋白质分子自由通过,是一种较为理想的分离纯化介质。
以木瓜蛋白酶为模型蛋白,采用静态吸附法和动态脱附法研究染料亲和膜色谱介质的蛋白质吸附性能。静态吸附法考察了配基供给量、缓冲液pH值、缓冲液钠离子强度、吸附温度和吸附时间等对木瓜蛋白酶吸附量的影响。在吸附条件为：染料配基浓度10.0mg/mL、缓冲液pH8.5、钠离子强度为0M、吸附温度为37℃、吸附3h时最大静态吸附量达到37.0mg/g。动态脱附法考察了洗脱液、洗脱液pH、洗脱液钠离子强度和洗脱速度等对木瓜蛋白酶脱附量的影响。在动态脱附条件为NaCl洗脱液pH6.0,钠离子强度为1.0M,洗脱速度为1mL/min地洗脱下,达到最大的动态脱附效果,纯化后的酶活力达到1035.53U/mg,纯化倍数约为20倍。
采用吸附等温模型、吸附动力学模型、吸附热力学模型和计算机模拟实验阐述染料亲和膜对模型蛋白木瓜蛋白酶的吸附机理。以Langmuir吸附模型和Freundlich吸附模型考察染料亲和膜的蛋白质吸附行为。研究发现Freundlich吸附模型可以较好地解释染料亲和膜的吸附行为,证实亲和膜对木瓜蛋白酶的吸附是不均匀立体结构的多分子层吸附模型。吸附动力学模型分别用准一级动力学吸附模型、准二级动力学吸附模型、粒子内扩散吸附模型以及Elovich动力学吸附模型考察染料亲和膜的吸附行为。研究发现准二级动力学吸附模型可以较好解释该吸附行为,证实染料亲和膜表面染料配基的活性位点越多,吸附能力就越强,一旦染料配基的活性位点覆盖率变大,可供共价结合的位点减少,吸附能力下降。吸附热力学研究结果为：焓变值ΔH0、熵变值ΔS0、吉布斯自由能ΔG0分别为18.52kJ/mol,0.077kJ/(mol K)和-4.43kJ/mol(298K)o焓变值ΔH0为18.52kJ/mol表明该吸附过程为吸热反应,温度的增加有利于吸附行为的发生,吸附作用力包括化学作用和物理作用。熵变值ΔS0为0.077kJ/(mol K)表明吸附过程是一个染料配基表面先脱附水分子,再吸附蛋白分子的循环过程,即“溶剂置换作用”,同时证实整个体系的吸附过程是混乱度增加的吸热过程。吉布斯自由能AG为-4.43kJ/mol(298K)不仅证明了吸附行为是自发的,还证实物理作用与化学作用同样存在于该吸附行为中,并发挥主要作用。采用Maestro9.0软件对染料配基和木瓜蛋白酶进行分子对接计算机模拟实验,结果证实吸附行为不仅包含物理吸附(疏水作用),还包括化学偶联作用(氢键)。
染料Reactive Red120亲和膜初步应用于木瓜蛋白酶的分离纯化。通过上样,分离,洗涤,洗脱四个步骤,收集酶活最高的一组组分,冷冻干燥后经比酶活计算和FPLC表征,得到纯化倍数为粗酶溶液22.60倍的木瓜蛋白酶粉,证实所制备的染料Reactive Red120亲和膜能有效的用于木瓜蛋白酶的富集和分离,能满足皮革、饲料等领域对中等纯度的木瓜蛋白酶需求。
论文同时以木瓜蛋白酶为模型,通过噬菌体展示技术筛选的七肽(Ile-Gln-Ser-Pro-His-Phe-Phe)为配基,以戊二醛为交联剂,以交联噬菌体为载体,利用生物法制备出交联噬菌体七肽亲和吸附介质(CAPOP)。通过透射电镜、动态光散射仪、酶联免疫吸附检测对CAPOP进行定性分析,确定七肽配基的ELISAO0492值为1.9,证实对木瓜蛋白酶具有亲和作用,交联噬菌体团聚物的粒径为5-6μm,是单个噬菌体粒径的10倍左右,证实交联成功。该介质具备特异性吸附高,价格低廉,制备简易的优点。
采用静态吸附法、响应面优化法和动态脱附法研究CAPOP的蛋白吸附性能。静态吸附法主要考察了缓冲液pH值、缓冲液钠离子强度、吸附温度和吸附时间等对木瓜蛋白酶吸附量的影响。在吸附条件为缓冲液pH7.0,钠离子强度为0M,吸附温度为25℃,吸附2h时最大静态吸附量达到50mg/g。采用响应面法分析了吸附过程中多种因素共作用对木瓜蛋白酶吸附量的关系。研究发现不同因素的共作用对吸附量有显著影响,得到回归方程并建立优化条件：噬菌体展示配基含量为1011PFU/mL,pH值为6.9,温度为32℃,木瓜蛋白酶粗酶溶液浓度为7.51mg/mL,木瓜蛋白酶的吸附量最高,为55.4229mg/g。动态脱附法考察了洗脱液,洗脱液pH、洗脱液钠离子强度和洗脱速度对木瓜蛋白酶吸附量的影响。在动态脱附条件为pH9.0,钠离子强度为1.0M,洗脱速度为1.0mL/min的NaSCN溶液的洗脱下,达到最大的动态脱附效果,纯化倍数达200倍左右,纯化后的酶活力达到4647.73U/mg,基本与商品酶酶活持平。
采用吸附等温线模型,吸附动力学模型,吸附热力学模型和计算机模拟技术探讨交联噬菌体对木瓜蛋白酶的吸附机理。分别以Langmuir吸附模型和Freundlich吸附模型考察CAPOP的吸附行为。研究发现Freundlich吸附模型可以较好地解释CAPOP的吸附行为,证实CAPOP对木瓜蛋白酶的吸附是不均匀立体结构的多分子层吸附模型,这与交联噬菌体的立体结构相吻合。吸附动力学模型分别以准一级动力学吸附模型、准二级动力学吸附模型和Elovich动力学吸附模型考察CAPOP的吸附行为。研究发现准二级动力学吸附模型可以解释该吸附行为,证实CAPOP表面展示的七肽配基的活性位点越多,吸附能力就越强,一旦七肽配基的活性位点覆盖率变大,可供共价结合的位点减少,吸附能力下降。吸附热力学研究表明：温度在25℃以下,焓变值ΔH0为7.97kJ/mol说明吸附反应为吸热反应,温度地升高有利于吸附行为地发生,作用力包括化学作用和物理作用。ΔS0为0.044kJ/(mol K)表明吸附过程是一个七肽配基表面先脱附水分子,再吸附蛋白分子的循环过程,即“溶剂置换作用”,同时证实整个体系的吸附过程是混乱度增加的吸热过程。吉布斯自由能ΔG0为-5.14kJ/mol(298K)不仅证明了吸附行为是自发的,还证实物理作用与化学作用同样存在于该吸附行为中,并发挥主要作用。在25℃以上,焓变值ΔH0为-12.02kJ/mol,熵变值ΔS0为-0.023kJ/(mol K),熵减焓减,说明吸附体系存在“转变温度”,这可能是由于吸附过程产生了新的化学键。但吉布斯自由能ΔG0为-5.17kJ/mol (298K),仍证明吸附行为是自发的,说明吸附过程存在物理作用与化学作用,并随温度的升高产生了新的化学键。采用Maestro9.0软件对七肽配基和木瓜蛋白酶进行分子对接计算机模拟实验,结果证实吸附行为不仅包含物理吸附(疏水作用),还包括化学偶联作用(氢键作用)。
CAPOP初步应用于木瓜蛋白酶的分离纯化。通过亲和吸附四个步骤,收集酶活最高的组分,冷冻干燥后经比酶活的计算和SDS-PAGE表征,得到纯化倍数为粗酶溶液的240.94倍,是染料亲和膜的近10倍。证实所制备的CAPOP不仅能有效的分离纯化木瓜蛋白酶,并表现为更高的分离效率,具备满足医疗、生物生化工程、抗体工程等领域对高纯度木瓜蛋白酶需求的潜力。
以木瓜蛋白酶为模型蛋白,研究七肽配基对木瓜蛋白酶的抑制行为和抑制机理。通过考察不同浓度的七肽分子对木瓜蛋白酶催化活性的抑制效果,采用Lineweaver-Burk双倒数作图,最终确定当催化体系中七肽分子的浓度达到20mM时,相对抑制率达到了66.41%,得出导致木瓜蛋白酶酶活丧失50%时的七肽分子浓度(IC50)为14.2mM；当七肽分子浓度为20mM时,催化体系的延滞时间为421s,延长了155%。同时证明七肽分子对木瓜蛋白酶酶活的抑制作用是属于可逆性抑制。
综上所述,本论文分别通过化学法和生物法制备两种新型的亲和色谱介质,设计思路具有典型性和拓展性,可推广到任一目标分离物(主要是蛋白质)的特异性配基筛选和规模性纯化,这为新型生物多肽配基的开发、亲和色谱分离过程研究、分离工艺流程选择和设计提供了相关基础数据和理论指导,从而拓宽了亲和色谱技术的应用范围。
Affinity chromatography is a technology of separation and purification, by taking advantage of specific affinity between biological macromolecules and the solid phase surface. With the development of high-performance affinity chromatography, it has been applied in organic chemistry, biochemistry, molecular biology, bioengineering, genetic engineering and other fields. However, due to its expensive affinity ligands, high operating pressure, small handling capacity and other shortcomings, it is difficult to play a much more significant role under the premise of low-cost and high efficiency. To overcome this limit, the developments of the small molecules similar to affinity ligands and new low-cost carrier materials have been widely investigated. More and more scholars are pay attention on screening ligands through a combination method and improving the existing materials, but the former method greatly increases the workload and the cost, and the latter method is unable to overcome the dilemma of complicated operation and small handling capacity. There are good solutions to these problems by adopting dye and metal ion as affinity ligand, or screening for the ligands by biopanning, which could separate the targets proteins rapidly and efficiently and keep the targets high activity. In this regards, the dye and biomimetic affinity chromatography are be concerned by lots of scholars.
Using papain as model protein, two affinity chromatography medium were prepared by chemical and biological methods:dye affinity membrane and cross-linking affinity phage oligopeptide pellet. Adsorption capacity and adsorption mechanism had been studied and the effectiveness of the separation and purification of model protein had been evaluated.
Firstly, using nylon membranes with good mechanical strength as the carrier, covalent coupling of chitosan (CS) to activated nylon membrane was performed after the reaction of the microporous nylon membrane with formaldehyde. The dye Reactive Red120as a ligand was then covalently immobilized on the CS-coated membranes. The dye affinity membranes were characterizied by SEM, UV spectroscopy, FT-IR and elemental analysis. The results showed the chitosan content of dye affinity membranes was determined of118.5±7.5mg/g and Reactive dye Reactive Red120was successfully bond to the modified nylon membrane with the load capacity of163.4±10.3μmol/g. SEM showed the pore sizes of affinity membrane were distributed in the range of about0.5-3.0μm, which facilitated mass transfer. So the dye affinity membrane could be used as an ideal separation and purification media.
The protein adsorption behavior and capacity of the new dye affinity membrane absorbent were studied by static and dynamic method, using papain as a model protein. By testing the different concentrations of ligand, pH, Na ionic strength, temperature and adsorption time, a nice result was achieved:at the circumstances of pH8.5,0M Na ionic strength,37℃, maximum static adsorption of papain was reached to37mg/g after3h. Dynamic testing included elution rate, pH, Na ionic strength and other factors. It showed in the condition of pH6.0,1M ionic strength, elution rate of1mL/min, the papain with the highest activity (1035.53U/mg) would be eluted with20-fold purification.
Adsorption mechanisms of the dye affinity membrane were described by adoption of adsorption isotherm model, adsorption kinetics model, adsorption thennodynamic model and computer simulation study. Adsorption isotherm analysis was identified by Langmuir model and Freundlich model. The linear fit of Freundlich model indicating that the adsorption type is a3-dimensional structure of multi-molecular layer adsorption model. The adsorption kinetics models studies were combined by Pseudo-first-order kinetic model, Pseudo-second-order kinetic model, Intra-particle diffusion model and the Elovich kinetic model. It was found that the Pseudo-second-order kinetic model can better explain the adsorption behavior, indicating that the adsorption capacity was always proportionate to the amounts of active sites on dye affinity membrane surface, once the active sites covered, the adsorption capacity decreased. The values of the standard enthalpy changes, the entropy changes and Gibbs free energy were18.52kJ/mol,0.077kJ/(mol. K),-4.43kJ/mol (298K), respectively. ΔH0value of18.52kJ/mol indicates that the adsorption process is an endothermic reaction, the adsorption capacity increased when temperature elevated and the adsorption force including chemical and physical effects. ΔS0value of0.077kJ/(mol. K) showed the desorption of water molecules occured before adsorption of papain, which named "solvent replacement role", and also confirmed the adsorption process of the system increased disorder. ΔG0value of-4.43kJ/mol (298K) proved that the adsorption behavior was spontaneous, and confirmed that the physical and chemical effects also exist in the adsorption behavior. Computer simulations using the Maestro9.0software also confirmed both the existing of physical (hydrophobic interactions) and chemical effects (hydrogen bonding).
Papain as a model was purified by dye Reactive Red120affinity membrane. Papain was purified22.6-fold in a single step using Red120-CS-membrane, as determined by protein content and enzyme activity assays, meeting papain demands of leather, feed and other areas.
Using7-mer peptides (Ile-Gln-Ser-Pro-His-Phe-Phe) from phage display library as ligand, glutaraldehyde as crosslinking agent and cross-linked phage as matrix, cross-linking affinity phage-oligopeptide pellet (CAPOP) was prepared. Qualitative analysis of enzyme-linked immunosorbent assay confirmed the7-mer peptides ligand have an affinity for papain with OD492value of1.9. Particle size of CAPOP was determined5-6μm by DLS and TEM, indicating the cross-linking successfully. The CAPOP absorbent have the advantages of specific adsorption, low cost and simple preparation.
The adsorption capacity of CAPOP was studied by static adsorption, response surface methodology and dynamic desorption. The static adsorption mainly concerned on the buffer pH, Na ionic strength, the adsorption temperature and adsorption time. The maximum static adsorption capacity reached50mg/g with the optimization of conditions of pH7.0, Na ionic strength0M,25℃and2h. The RSM analysed the relationship between a variety of factors and adsorption capacity as well as establish a model. The maximum papain adsorption capacity reached55.4229mg/g under the conditions of the phage display amount of ligand1011PFU/mL, pH6.9,32℃and papain crude enzyme solution concentration7.51mg/mL. The dynamic desorption concerned about type, pH, Na ionic strength of eluent and the elution speed. A purification of200-fold with its enzyme activity4647.73U/mg was achieved under the dynamic desorption conditions of NaSCN, pH9.0, Na ionic strength1.0M, the elution speed1.0mL/min.
Adsorption mechanisms of CAPOP were studied by adoption of adsorption isotherm model, adsorption kinetics model, adsorption thermodynamic model and computer simulation study. Adsorption isotherm analysis was identified by Langmuir model and Freundlich model. The study showed that the Freundlich model could better explain the adsorption behavior, indicating the adsorption type was a3-dimensional structure of multi-molecular layer adsorption model, which was consistent with the structure of cross-linked phage. The adsorption kinetics models studies were analysed by Pseudo-first-order kinetic model, Pseudo-second-order kinetic model and the Elovich kinetic model. Results showed that the Pseudo-second-order kinetic model could better explain the adsorption behavior, indicating that the adsorption capacity was always proportionate to the amounts of active sites on CAPOP. The values of the standard enthalpy changes, the entropy changes and Gibbs free energy were7.97kJ/mol,0.044kJ/(mol. K),-5.14kJ/mol (298K), respectively. ΔS0value of0.044kJ/(mol. K) showed that the desorption of water molecules occurs before adsorption of papain, which named "solvent replacement role", and also confirmed the adsorption process of the system increased disorder. ΔG0value of-5.14kJ/mol (298K) proved the adsorption behavior was spontaneous, and confirmed that the physical and chemical effects also exist in the adsorption behavior and play a major role. Above25℃, ΔH0value of-12.02kJ/mol, ΔS0value of-0.023kJ/(mol K), indicated that there was a "transition temperature" in adsorption system, which may be due to the new chemical bonds in adsorption process. But ΔG0value of-5.17kJ/mol (298K) still proved the adsorption behavior was spontaneous. Computer simulations using the Maestro9.0software also confirmed both the existing of physical (hydrophobic interactions) and chemical effects (hydrogen bonding).
Papain was purified by CAPOP. Papain was purified240.94-fold in a single step using CAPOP, as determined by protein content and enzyme activity assays. It was confirmed CAPOP was an effective separation and purification absorbents with higher separation efficiency, which have the potential to meet the demands of medical, biological and biochemical engineering, antibody engineering and other fields.
Inhibition mechanism and inhibition behavior of7-mer peptide affinity ligands for papain was studied. Finally it was confirmed the delay time was increasing with the concentration of7-mer peptide increasing, and the7-mer peptide molecule concentration (IC50) was14.2mM by Lineweaver-Burk double reciprocal model. When the catalytic system didn't contain7-mer peptide molecules, the enzyme reaction delay time was165s, whiled the lag time was421s when the concentration of7-mer peptide molecular came to20mM. The results also showed that with the increasing of the7-mer peptide molecules concentration, the catalysis efficiency decreased, indicating that the activity was inhibited by the7-mer peptide molecules, rather than by reducing the amount of enzyme.7-mer peptide molecules on the activity of papain inhibition were identified as irreversible inhibition process.
In summary, we prepared two kinds of affinity chromatography absorbents by chemical and biological methods. It could be expanded any other field of protein separation process including ligand screening, matrix preparation and the design of separation process. Through the study of this thesis, we wish to prodive fundamental data and theory guidence for the application of affinity chromatograph.
引文
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