亲水性聚合物在基因载体、表面蛋白固定及蛋白结晶方面的应用
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摘要
原子转移自由基聚合(ATRP)自1995年诞生以来,发展速度惊人,不仅成为当前最重要的“活性”/可控聚合方法,而且已经延伸到各种功能材料领域中。在生物材料领域也得到广泛应用,主要包括生物材料表面改性和生物大分子载体两方面。多肽/蛋白类药物和基因治疗的前景诱人,但对其载体的要求很高,多才多艺的ATRP技术很有可能就是合成其载体的最佳方法。
本论文应用ATRP的聚合方法合成了一系列亲水性聚合物,并从聚合物基因载体、固定蛋白质的硅片表面的改性以及溶菌酶蛋白结晶沉淀剂三方面讨论了合成聚合物的结构对于基因转染效率、硅片表面的蛋白质分子固定以及溶菌酶蛋白结晶的影响。
随着基因治疗研究的不断深入,选择更安全有效的基因转移载体已成为基因治疗成功的关键。病毒载体基因转移效率很高但其主要缺点是具有免疫原性,使用的安全性差；一些合成载体虽然比重组病毒载体更安全,但基因转移效率不能达到要求。近几年合成了一系列专门为基因转移而设计的聚合物基因载体,并研究了它们结构一功能的相互关系。随着对聚合物基因转移机理的更深入的理解和对现有聚合物载体更进一步的完善,聚合物基因转移体系将成为人类基因治疗的有力工具。
论文首先研究了嵌段顺序对聚合物基因载体的影响。选用生物相容性好的甲基丙烯酸聚乙二醇单甲醚酯(PEGMA)和带有胺基的DMAEMA为单体,利用ATRP方法“活性”/可控和所合成聚合物分子量分布较窄的特点,合成了分子量相近且PDMAEMA嵌段含量相近,但嵌段结构不同的双嵌段共聚物PDMAEMA-b-poly(PEGMA)和三嵌段共聚物PDMAEMA-b-poly(PEGMA)-b-PDMAEMA。以这两种聚合物凝聚质粒DNA分子,用琼脂糖凝胶电泳、原子力显微镜(AFM)及动力学光散射(DLS)等表征方法分析DNA分子凝聚的形态和大小,并进行了293T细胞的转染和细胞毒性实验。结果表明,尽管两种嵌段共聚物所带电荷相同,不同的嵌段结构导致了所形成的阳离子聚合物/DNA复合物,即使在相同N/P比的情况下,在溶液中的组装结构和所带表面电荷也有所不同,并导致了对293T细胞系的转染效率和毒性有较大的差异。
然后进一步讨论了聚合物的胺基密度对聚合物基因载体的影响。利用ATRP方法可以在单体上连接功能基团的特点,实验中设计合成了胺基密度不同的多胺类聚合物PBAMAM、PBAMAM-b-poly(PEGMA)和PBAMAM-b-PDMAEMA。以这三种聚合物凝聚质粒DNA分子,用琼脂糖凝胶电泳和原子力显微镜(AFM)进行了观测并进行了293T细胞的转染实验。实验证明适当的胺基密度对聚合物基因载体的性能十分重要。
蛋白质微阵列分析中的一个研究热点是基底表面固定蛋白质分子的能力,所固定的蛋白质分子既保持本身的构象又保持原有的活性位点。利用共价连接或非共价亲和结合,有一些技术已被用来固定蛋白质分子和其他生物分子。多数情况下这些固定方法是非特异性的,所固定的蛋白质分子在基底表面是随机取向的。ATRP方法能在生物材料表面生长一层致密的分子刷,从而改变生物材料原有的表面性质。将分子刷末端修饰上功能基团,可以实现基底表面对蛋白质分子的共价偶合固定。共价偶合固定蛋白是制备蛋白质微阵列的重要方法,也有较多优点：(1)与物理吸附相比,共价结合使蛋白质分子与基底表面结合得更牢固,检测信号更强；(2)共价偶合的方法可以有效的降低蛋白质分子对基底的非特异性吸附；(3)分析物范围较宽,可以是小分子也可以是生物大分子或细胞器等。
实验中在硅片表面合成了poly(PEGMA)分子刷,并利用ATRP方法可以在聚合物末端连接功能基团的特点,在分子刷末端用NHS基团修饰,得到了Si-poly(PEGMA)-NHS表面。通过X射线光电子能谱(XPS)、AFM和扫描电子显微镜(SEM)等方法对硅片的每步修饰进行了表征,并对实验中出现的三种表面：Si-poly(PEGMA)、Si-poly(PEGMA)-amine和Si-poly(PEGMA)-NHS固定蛋白质分子的效果进行了比较。前两种表面分别是通过物理吸附和静电作用固定蛋白分子的。我们发现通过共价键与蛋白分子连接的Si-poly(PEGMA)-NHS表面对蛋白分子的固定效果最好。尽管这种修饰方法仍存在不足之处,但为共价固定蛋白分子提供了一种新的思路。
蛋白质等生物大分子是生物活性物质中非常重要的一类,他们在医学、药学、化学等领域具有很高的应用价值,因此对蛋白质结构的研究就显得尤为重要。而在目前来说只有通过获得适当的蛋白晶体、进而通过X射线衍射才是确定蛋白质空间三维结构最可靠的办法,因为以晶体形式存在的蛋白质是最稳定的。蛋白质分子的结晶是一个多参数控制的复杂过程,包括物理、化学、生物化学等因素,具体的分为：温度、时间、电解质性质、粘度、离子强度、过饱和度、蛋白质纯度、蛋白质本身的对称性以及蛋白质稳定性和蛋白质等电点等因素的影响。为了获得合适的蛋白晶体就必须对这些因素加以考虑,通过选择合适的沉淀剂来提高蛋白的结晶效率。
近年来,在生物矿化领域的研究中,嵌段共聚物通过自身在溶液中的自组装来调控碳酸钙等小分子物质在溶液中的结晶形态,进而获得高性能的复杂形貌和多级结构的复合材料受到了广泛的关注。这些形态的形成机制主要集中在聚合物的选择性吸附、中尺度转化以及高尺度组装等方面。通过探讨嵌段共聚物在溶液中的自组装行为是否对蛋白质分子的结晶有影响,可以进一步拓宽蛋白质结晶沉淀剂的选择范围。
实验中合成了一系列聚合物,以这些聚合物作为溶菌酶蛋白结晶的沉淀剂。利用小角X射线散射(SAXS)、光散射(DLS & SLS)以及透射电镜(TEM)等方法探讨了嵌段共聚物在溶液中的自组装行为,并观察了嵌段共聚物的结构、浓度对溶菌酶晶体成核、晶体生长以及晶体形态的影响。通过实验证明,含有PDMAEMA嵌段的聚合物有利于溶菌酶的结晶,聚合物的自组装形态对蛋白分子的成核和晶体形态有很大影响。当聚合物溶液中加入大量电解质小分子(tascimate盐溶液)时,高离子强度使聚合物的自组装遭到一定程度的破坏,最后形成的溶菌酶蛋白晶体形貌没有明显的差别。相反,从溶液中去掉这些小分子化合物后,在嵌段共聚物的作用下溶菌酶晶体的晶核数量、晶体大小及晶体形貌都产生了变化。
简言之,本博士论文以亲水性聚合物为研究对象,探讨了亲水性聚合物在基因治疗、蛋白质芯片以及蛋白质结晶三方面的应用。
The development of Atom Transfer Radical Polymerization (ATRP) was incredible since its emergence in 1995. ATRP is one of the most important controlled/"living" polymerization techniques and enables preparation of different functional materials. In the biological field, ATRP has been proposed for the polymerization of polymer brushes to modify the surface of biological material, and of biomacromolecular vectors. Especially for polypeptide/protein medicines and gene therapy, the successful design of multifunctional vectors is mostly suitable for ATRP.
In this dissertation we synthesized a series of hydrophilic polymers by ATRP. The effects of polymer structures on gene transfection efficiency, protein immobilization on silicon surface and lysozyme crystallization were evaluated.
As the concept of gene therapy expanded, the choice of safer and more efficient vectors for delivery of genes is the key to successful gene therapy. Viral vectors are typically very efficient, but the main drawback related to viral vectors is the safety concerns. Synthetic gene-delivery vectors, although safer than recombinant viruses, generally do not possess the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure-function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.
First we studied the influence of block sequence of block copolymers on the efficacy of polymer gene vectors. PEGMA, of good biocompatibility and DMAEMA with two amine groups were polymerized by ATRP for its feature of controlled/"living" and low polydispersity, to give the diblock copolymer PDMAEMA-b-poly(PEGMA) and triblock copolymer PDMAEMA-b-poly(PEGMA)-b-PDMAEMA with same molecular weights, same PDMAEMA content and different block sequences. The two block copolymers were used to induce DNA condensation, investigated by agarose gel electrophoresis, atomic force microscopy (AFM), dynamic light scattering (DLS), transfection of 293T cell line and MTT cytotoxicity assay. Significant differences on the assembling configuration and surface charge of polycation/DNA complexes were observed due to block sequences with same N/P ratios, and consequently different 293T cell transfection efficacy and cytotoxicity were obtained.
Then the effects of amine group density of polymeric transfectants were evaluated. Polyamines PBAMAM, PBAMAM-b-poly(PEGMA) and PBAMAM-b-PDMAEMA with different amine group densities were synthesized by ATRP for the modification of monomers and studied by agarose gel electrophoresis, AFM and 293T cell transfection. As a result, optimal amine group density is of great importance for the properties of polymeric transfectants.
A key focus in protein microarray analysis is the ability to immobilize proteins in their native conformation on substrate surfaces while preserving active sites for functional studies. Several approaches have been developed to immobilize proteins and other biomolecules onto substrate surfaces, using either covalent attachment or noncovalent affinity binding chemistries. In most cases, however, these modes of immobilization are nonspecific, causing the molecules to be randomly orientated on the sufaces. Polymer brushes can be densely prepared by ATRP on biomaterial surfaces, and introduce new properties to the surfaces. Covalent coupling for protein immobilization is an important strategy, and has many advantages:(1) compared with physical adsorption, covalent coupling provides more efficient binding to surfaces and stronger detection signals; (2) covalent coupling is often combined with the protection of the substrate to prevent nonspecific adsorption; (3) covalent coupling allows access to the immobilized protein molecules for small analytes such as ligands and larger analytes such as biomacromolecules or organelles.
In the experiment, poly(PEGMA) brushes were synthesized by ATRP to modify the ends of each polymer chain with NHS groups, to give the final surfaces Si-poly(PEGMA)-NHS. After each modification step, the surfaces were characterized by X-ray photoelectron spectroscopy (XPS), AFM and SEM. We compared the ability for protein immobilization of three kinds of silicon surfaces encounter in the process of synthesis:Si-poly(PEGMA), Si-poly(PEGMA)-amine and Si-poly(PEGMA)-NHS. Si-poly(PEGMA) immobilizes proteins by physical adsorption and Si-poly(PEGMA)-amine immobilizes by electrostatic interaction. Si-poly(PEGMA)-NHS which immobilizing proteins by covalent coupling has the greatest performance. Although this modification process has some disadvantages, it provide a new way to synthesize covalent binding surfaces.
As we know, protein is a significantly important part of bioactive matters and the targets that most of the medicaments acting on in medical, biology and chemistry fields. So it is becoming significantly important to determine the three dimension structures of proteins. To obtain protein crystals and through the X-ray diffraction is the main route toward structure determination of protein. Crystallization of protein molecules is a multiparameter controlled and complicated process including physical, chemical and biological factors. These parameters are concretely including temperature, time, properties of electrolytes, viscosity, ionic strength, super saturation, purity of proteins, symmetry and stability of protein structure, isoelectric point and so on. To acquire the suitable crystals all the parameters must be taken into account, and choose the best precipitator to improve the crystallization of protein molecule.
In biomineralization research field, crystal morphology control of inorganic minerals such as calcium carbonate by using self-assembly of block copolymers, to obtain composite materials with high-performance pattern and hierarchical structures are showed great interest in recent years. These different morphogenesis mechanisms mainly focus on selective polymer adsorption, mesoscopic transformations and higher order assembly. By discussion of the effects of self-assembly of block copolymers on crystallization of protein molecules, it was found that block copolymers can serve as more efficient precipitator candidates in protein crystallization.
Herein we prepared a series of polymers, and used these polymers as precipitators in lysozyme crystallization to investigate there self-assembly in solution and the effects of polymers on the morphology of protein crystals. Small Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS), Static Light Scattering (SLS) and Transmission Electron Micrograph (TEM) methods were used to investigate the self-assembly of polymers in solution. The effects of the structures and concentration of polymeric precipitators on the lysozyme nucleation, crystal growth and crystal morphology were observed. We found that polymers with PDMAEMA blocks are favorable for nucleation and the self-assembling morphology of polymers significantly affect lysozyme nucleation and crystal growth. The presence of high ionic strength (abundant electrolytes, tacsimate solution) in the mother liquor could destroy the self-assembling processes of polymers, and thus the final morphology of lysozyme crystals did not have significant differences. In contrast, when the tacsimate was excluded from the solutions, different amount of crystal nucleus, crystal sizes and crystal morphologies of lysozyme crystals appeared.
In brief, in this doctoral dissertation, hydrophilic polymers were focused on and investigated on their application in gene therapy, protein microarrays and protein crystallization.
本论文应用ATRP的聚合方法合成了一系列亲水性聚合物,并从聚合物基因载体、固定蛋白质的硅片表面的改性以及溶菌酶蛋白结晶沉淀剂三方面讨论了合成聚合物的结构对于基因转染效率、硅片表面的蛋白质分子固定以及溶菌酶蛋白结晶的影响。
随着基因治疗研究的不断深入,选择更安全有效的基因转移载体已成为基因治疗成功的关键。病毒载体基因转移效率很高但其主要缺点是具有免疫原性,使用的安全性差；一些合成载体虽然比重组病毒载体更安全,但基因转移效率不能达到要求。近几年合成了一系列专门为基因转移而设计的聚合物基因载体,并研究了它们结构一功能的相互关系。随着对聚合物基因转移机理的更深入的理解和对现有聚合物载体更进一步的完善,聚合物基因转移体系将成为人类基因治疗的有力工具。
论文首先研究了嵌段顺序对聚合物基因载体的影响。选用生物相容性好的甲基丙烯酸聚乙二醇单甲醚酯(PEGMA)和带有胺基的DMAEMA为单体,利用ATRP方法“活性”/可控和所合成聚合物分子量分布较窄的特点,合成了分子量相近且PDMAEMA嵌段含量相近,但嵌段结构不同的双嵌段共聚物PDMAEMA-b-poly(PEGMA)和三嵌段共聚物PDMAEMA-b-poly(PEGMA)-b-PDMAEMA。以这两种聚合物凝聚质粒DNA分子,用琼脂糖凝胶电泳、原子力显微镜(AFM)及动力学光散射(DLS)等表征方法分析DNA分子凝聚的形态和大小,并进行了293T细胞的转染和细胞毒性实验。结果表明,尽管两种嵌段共聚物所带电荷相同,不同的嵌段结构导致了所形成的阳离子聚合物/DNA复合物,即使在相同N/P比的情况下,在溶液中的组装结构和所带表面电荷也有所不同,并导致了对293T细胞系的转染效率和毒性有较大的差异。
然后进一步讨论了聚合物的胺基密度对聚合物基因载体的影响。利用ATRP方法可以在单体上连接功能基团的特点,实验中设计合成了胺基密度不同的多胺类聚合物PBAMAM、PBAMAM-b-poly(PEGMA)和PBAMAM-b-PDMAEMA。以这三种聚合物凝聚质粒DNA分子,用琼脂糖凝胶电泳和原子力显微镜(AFM)进行了观测并进行了293T细胞的转染实验。实验证明适当的胺基密度对聚合物基因载体的性能十分重要。
蛋白质微阵列分析中的一个研究热点是基底表面固定蛋白质分子的能力,所固定的蛋白质分子既保持本身的构象又保持原有的活性位点。利用共价连接或非共价亲和结合,有一些技术已被用来固定蛋白质分子和其他生物分子。多数情况下这些固定方法是非特异性的,所固定的蛋白质分子在基底表面是随机取向的。ATRP方法能在生物材料表面生长一层致密的分子刷,从而改变生物材料原有的表面性质。将分子刷末端修饰上功能基团,可以实现基底表面对蛋白质分子的共价偶合固定。共价偶合固定蛋白是制备蛋白质微阵列的重要方法,也有较多优点：(1)与物理吸附相比,共价结合使蛋白质分子与基底表面结合得更牢固,检测信号更强；(2)共价偶合的方法可以有效的降低蛋白质分子对基底的非特异性吸附；(3)分析物范围较宽,可以是小分子也可以是生物大分子或细胞器等。
实验中在硅片表面合成了poly(PEGMA)分子刷,并利用ATRP方法可以在聚合物末端连接功能基团的特点,在分子刷末端用NHS基团修饰,得到了Si-poly(PEGMA)-NHS表面。通过X射线光电子能谱(XPS)、AFM和扫描电子显微镜(SEM)等方法对硅片的每步修饰进行了表征,并对实验中出现的三种表面：Si-poly(PEGMA)、Si-poly(PEGMA)-amine和Si-poly(PEGMA)-NHS固定蛋白质分子的效果进行了比较。前两种表面分别是通过物理吸附和静电作用固定蛋白分子的。我们发现通过共价键与蛋白分子连接的Si-poly(PEGMA)-NHS表面对蛋白分子的固定效果最好。尽管这种修饰方法仍存在不足之处,但为共价固定蛋白分子提供了一种新的思路。
蛋白质等生物大分子是生物活性物质中非常重要的一类,他们在医学、药学、化学等领域具有很高的应用价值,因此对蛋白质结构的研究就显得尤为重要。而在目前来说只有通过获得适当的蛋白晶体、进而通过X射线衍射才是确定蛋白质空间三维结构最可靠的办法,因为以晶体形式存在的蛋白质是最稳定的。蛋白质分子的结晶是一个多参数控制的复杂过程,包括物理、化学、生物化学等因素,具体的分为：温度、时间、电解质性质、粘度、离子强度、过饱和度、蛋白质纯度、蛋白质本身的对称性以及蛋白质稳定性和蛋白质等电点等因素的影响。为了获得合适的蛋白晶体就必须对这些因素加以考虑,通过选择合适的沉淀剂来提高蛋白的结晶效率。
近年来,在生物矿化领域的研究中,嵌段共聚物通过自身在溶液中的自组装来调控碳酸钙等小分子物质在溶液中的结晶形态,进而获得高性能的复杂形貌和多级结构的复合材料受到了广泛的关注。这些形态的形成机制主要集中在聚合物的选择性吸附、中尺度转化以及高尺度组装等方面。通过探讨嵌段共聚物在溶液中的自组装行为是否对蛋白质分子的结晶有影响,可以进一步拓宽蛋白质结晶沉淀剂的选择范围。
实验中合成了一系列聚合物,以这些聚合物作为溶菌酶蛋白结晶的沉淀剂。利用小角X射线散射(SAXS)、光散射(DLS & SLS)以及透射电镜(TEM)等方法探讨了嵌段共聚物在溶液中的自组装行为,并观察了嵌段共聚物的结构、浓度对溶菌酶晶体成核、晶体生长以及晶体形态的影响。通过实验证明,含有PDMAEMA嵌段的聚合物有利于溶菌酶的结晶,聚合物的自组装形态对蛋白分子的成核和晶体形态有很大影响。当聚合物溶液中加入大量电解质小分子(tascimate盐溶液)时,高离子强度使聚合物的自组装遭到一定程度的破坏,最后形成的溶菌酶蛋白晶体形貌没有明显的差别。相反,从溶液中去掉这些小分子化合物后,在嵌段共聚物的作用下溶菌酶晶体的晶核数量、晶体大小及晶体形貌都产生了变化。
简言之,本博士论文以亲水性聚合物为研究对象,探讨了亲水性聚合物在基因治疗、蛋白质芯片以及蛋白质结晶三方面的应用。
The development of Atom Transfer Radical Polymerization (ATRP) was incredible since its emergence in 1995. ATRP is one of the most important controlled/"living" polymerization techniques and enables preparation of different functional materials. In the biological field, ATRP has been proposed for the polymerization of polymer brushes to modify the surface of biological material, and of biomacromolecular vectors. Especially for polypeptide/protein medicines and gene therapy, the successful design of multifunctional vectors is mostly suitable for ATRP.
In this dissertation we synthesized a series of hydrophilic polymers by ATRP. The effects of polymer structures on gene transfection efficiency, protein immobilization on silicon surface and lysozyme crystallization were evaluated.
As the concept of gene therapy expanded, the choice of safer and more efficient vectors for delivery of genes is the key to successful gene therapy. Viral vectors are typically very efficient, but the main drawback related to viral vectors is the safety concerns. Synthetic gene-delivery vectors, although safer than recombinant viruses, generally do not possess the required efficacy. In recent years, a variety of effective polymers have been designed specifically for gene delivery, and much has been learned about their structure-function relationships. With the growing understanding of polymer gene-delivery mechanisms and continued efforts of creative polymer chemists, it is likely that polymer-based gene-delivery systems will become an important tool for human gene therapy.
First we studied the influence of block sequence of block copolymers on the efficacy of polymer gene vectors. PEGMA, of good biocompatibility and DMAEMA with two amine groups were polymerized by ATRP for its feature of controlled/"living" and low polydispersity, to give the diblock copolymer PDMAEMA-b-poly(PEGMA) and triblock copolymer PDMAEMA-b-poly(PEGMA)-b-PDMAEMA with same molecular weights, same PDMAEMA content and different block sequences. The two block copolymers were used to induce DNA condensation, investigated by agarose gel electrophoresis, atomic force microscopy (AFM), dynamic light scattering (DLS), transfection of 293T cell line and MTT cytotoxicity assay. Significant differences on the assembling configuration and surface charge of polycation/DNA complexes were observed due to block sequences with same N/P ratios, and consequently different 293T cell transfection efficacy and cytotoxicity were obtained.
Then the effects of amine group density of polymeric transfectants were evaluated. Polyamines PBAMAM, PBAMAM-b-poly(PEGMA) and PBAMAM-b-PDMAEMA with different amine group densities were synthesized by ATRP for the modification of monomers and studied by agarose gel electrophoresis, AFM and 293T cell transfection. As a result, optimal amine group density is of great importance for the properties of polymeric transfectants.
A key focus in protein microarray analysis is the ability to immobilize proteins in their native conformation on substrate surfaces while preserving active sites for functional studies. Several approaches have been developed to immobilize proteins and other biomolecules onto substrate surfaces, using either covalent attachment or noncovalent affinity binding chemistries. In most cases, however, these modes of immobilization are nonspecific, causing the molecules to be randomly orientated on the sufaces. Polymer brushes can be densely prepared by ATRP on biomaterial surfaces, and introduce new properties to the surfaces. Covalent coupling for protein immobilization is an important strategy, and has many advantages:(1) compared with physical adsorption, covalent coupling provides more efficient binding to surfaces and stronger detection signals; (2) covalent coupling is often combined with the protection of the substrate to prevent nonspecific adsorption; (3) covalent coupling allows access to the immobilized protein molecules for small analytes such as ligands and larger analytes such as biomacromolecules or organelles.
In the experiment, poly(PEGMA) brushes were synthesized by ATRP to modify the ends of each polymer chain with NHS groups, to give the final surfaces Si-poly(PEGMA)-NHS. After each modification step, the surfaces were characterized by X-ray photoelectron spectroscopy (XPS), AFM and SEM. We compared the ability for protein immobilization of three kinds of silicon surfaces encounter in the process of synthesis:Si-poly(PEGMA), Si-poly(PEGMA)-amine and Si-poly(PEGMA)-NHS. Si-poly(PEGMA) immobilizes proteins by physical adsorption and Si-poly(PEGMA)-amine immobilizes by electrostatic interaction. Si-poly(PEGMA)-NHS which immobilizing proteins by covalent coupling has the greatest performance. Although this modification process has some disadvantages, it provide a new way to synthesize covalent binding surfaces.
As we know, protein is a significantly important part of bioactive matters and the targets that most of the medicaments acting on in medical, biology and chemistry fields. So it is becoming significantly important to determine the three dimension structures of proteins. To obtain protein crystals and through the X-ray diffraction is the main route toward structure determination of protein. Crystallization of protein molecules is a multiparameter controlled and complicated process including physical, chemical and biological factors. These parameters are concretely including temperature, time, properties of electrolytes, viscosity, ionic strength, super saturation, purity of proteins, symmetry and stability of protein structure, isoelectric point and so on. To acquire the suitable crystals all the parameters must be taken into account, and choose the best precipitator to improve the crystallization of protein molecule.
In biomineralization research field, crystal morphology control of inorganic minerals such as calcium carbonate by using self-assembly of block copolymers, to obtain composite materials with high-performance pattern and hierarchical structures are showed great interest in recent years. These different morphogenesis mechanisms mainly focus on selective polymer adsorption, mesoscopic transformations and higher order assembly. By discussion of the effects of self-assembly of block copolymers on crystallization of protein molecules, it was found that block copolymers can serve as more efficient precipitator candidates in protein crystallization.
Herein we prepared a series of polymers, and used these polymers as precipitators in lysozyme crystallization to investigate there self-assembly in solution and the effects of polymers on the morphology of protein crystals. Small Angle X-ray Scattering (SAXS), Dynamic Light Scattering (DLS), Static Light Scattering (SLS) and Transmission Electron Micrograph (TEM) methods were used to investigate the self-assembly of polymers in solution. The effects of the structures and concentration of polymeric precipitators on the lysozyme nucleation, crystal growth and crystal morphology were observed. We found that polymers with PDMAEMA blocks are favorable for nucleation and the self-assembling morphology of polymers significantly affect lysozyme nucleation and crystal growth. The presence of high ionic strength (abundant electrolytes, tacsimate solution) in the mother liquor could destroy the self-assembling processes of polymers, and thus the final morphology of lysozyme crystals did not have significant differences. In contrast, when the tacsimate was excluded from the solutions, different amount of crystal nucleus, crystal sizes and crystal morphologies of lysozyme crystals appeared.
In brief, in this doctoral dissertation, hydrophilic polymers were focused on and investigated on their application in gene therapy, protein microarrays and protein crystallization.
引文
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