基因工程仿生蜘蛛丝蛋白中重复模块对成丝性质的影响
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摘要
蜘蛛丝作为一种优越的蛋白质材料已经被发现可以应用于航天航空,生物医学工程,军工防弹衣等方面,但是由于蜘蛛的互相残杀习性,蜘蛛丝的获得不能通过类似于家蚕养殖的简单方式大量获得,20世纪末开始有科学家尝试在山羊、植物、昆虫等细胞中表达蜘蛛丝的基因,但是目前还是没有办法解决蜘蛛丝全长基因表达和蛋白产率低的问题。
蜘蛛丝的基因主要由N端、C端非重复序列和中间的大量重复序列组成,目前对蜘蛛丝的研究主要分为：1,通过构建文库对不同种类蜘蛛不同类型蜘蛛丝全长基因的调取；2,研究蜘蛛丝蛋白成丝机理,主要涉及对N端、C端和中间序列在蜘蛛丝蛋白成丝过程中的作用,以及自组装过程中蛋白二级结构的变化；3,蜘蛛丝在生物医学工程方面的应用,主要涉及复合材料、药物缓释等内容。现在对蜘蛛丝的研究非常热,2010年连续在nature上发表了多篇关于蜘蛛丝成丝机理的研究论文。而且越来越多的人在关注蜘蛛丝在应用方面的研究,如Thomas R. Scheibel利用蜘蛛丝蛋白成丝的自组装来选择性包装和释放药物,可以在不同溶液中形成胶囊,可应用于药物缓释等方面。将蜘蛛丝蛋白溶液与现有的组织工程材料复合成新型的材料,期望得到更好组织相容性并可降解的材料,在替代性皮肤和手术缝合线方面会有很好的应用。
目前已经有很多文献报道蜘蛛丝基因表达的N端非重复模块可能构成蜘蛛丝的分泌信号并且是pH敏感元件,但是更多的功能还没被发现,C端非重复模块可以控制蜘蛛丝蛋白的可溶性和纤维形成,在丝纤维自组装过程中都起到重要的作用,但是由于不同种类蜘蛛和不同蛛丝之间重复模块的同源性没有N端和C端高,重复模块分子量大、结构复杂,增加了对其研究的难度。重复模块在纤维形成的过程中的功能还有待进一步研究,本课题从分子水平和材料学水平上对不同数量重复模块成丝性质进行了研究。
我们实验室在前期试验中,通过基因文库筛选得到一段大约693bp的大腹园蛛基因序列,经过分析和比对我们初步确认其为含有完整重复单元的鞭毛状丝基因。对这段基因根据原核生物大肠杆菌密码子使用频率进行基因优化后,依据鞭毛状丝的特点将这段基因分别拼接得到分别含有1-4段重复序列的质粒PE-Sc1/2/3/4,在重复片段的两端分别加上人工合成的蛛丝基因的N端和C端基因。将拼接得到的四段基因与IMPACT (Intein-Mediated Purification with an Affinity Chitin-binding Tag)系统的PTWIN1质粒融合表达得到N-Sc1/2/3/4-C-Intein-CBD蛋白(质粒PT-N-Sc1/2/3/4-C),利用几丁质结合蛋白与几丁质树脂的特异性结合亲和层析得到N-Sc1/2/3/4-C-Intein-CBD蛋白,通过加入DTT诱导Intein发生N端断裂,得到N-Scl/2/3/4-C蛋白
将纯化的蛋白透析至pH7.0的去离子水中,利用六氟异丙醇(HFIP)溶剂将冷冻干燥后的蛋白按照20%的质量体积分数回溶后,通过湿纺的手段得到四种单纤维类蜘蛛丝。组成丝纤维的四种丝蛋白单分子内N端和C端非重复结构的数量是一样的,不同的是逐渐增加的重复模块数量。通过对四种单丝的应力应变测试发现随着重复模块数增多,丝纤维的延展性越好,最好的是含有四段重复模块的丝纤维,应变程度可以达到25%。但是纤维的抗张强度并不是随着蛋白质分子量增加一直增强,含有四段重复模块的丝纤维应力为60MPa,而含有三段重复模块的丝纤维应力达到了120MPa,是含有四段重复模块丝纤维的两倍,说明重复模块对丝纤维强度并不是主要影响因素。
再对四种单丝进行显微激光Raman测试,分析仿生蜘蛛丝的内部二级结构组成,通过研究发现四种重组蛛丝中,都含有α-helix、β-sheet、β-turn和random coil蛋白质二级结构。N端和C端非重复模块不是鞭毛状丝的基因(N端、C端和重复模块氨基酸序列见附录),主要组成α-螺旋、β-折叠和无规则卷曲结构。四种丝纤维在蛋白质二级结构上,其α-螺旋、β-折叠和无规则卷曲结构含量是大体一致的。鞭毛状丝重复模块氨基酸序列主要都是由GPGGX的结构组成,形成β-turn二级结构,在此基础上形成β-turn螺旋,该结构能增强丝纤维的弹性,再次证明了鞭毛状丝重复模块的增加可以增强仿生蛛丝的延展性。
本课题主要从蛋白水平和材料学水平研究重复模块在成丝过程中的重要作用,期望能够为获得性能优越的仿生蛛丝的提供研究基础,同时也对基因工程仿生蛛丝的应用提供一种新的思路。
As a kind of protein material, spider silk has excellent material properties that can be used in aerospace, biomedical engineering, military projects such as flak suit. Although spider silk cannot be produced by simply raising spider like silkworm because of spider internecine nature instinct, scientists have tried to express spider silk gene in goat, plant, insect cell after the end of 20th century. However, the difficulties of overall length gene expression problem and low protein productivity have not been solved nowadays.
The gene of spider silk is mainly composed of N non-repeat terminal, C non-repeat terminal and many repeat modules between N-terminal and C-terminal. At present, the research of spider silk focus on aspects as followed:1, Constructing library to retrieve the overall length of spider silks from different species and glands; 2, Understanding spider silk protein into fiber mechanisms, mainly referring to the functions of N-terminal, C-terminal and middle repeat sequences in the process spider silk proteins transfer into fibers, also including proteins the change of proteins secondary structure in self-assembly. 3, Application of spider silk in biomedical engineering relate in composite material, drug delivery and so on. At the present, research of spider silk become more and more hot. Many research papers were continuously published in 2010 Nature magazine related the mechanism of spider silk protein transferring to protein fiber. More and more people pay close attention to application area of spider silk. Thomas R. Scheibel selectively embeded and released drug by self-assemble character of spider silk protein, formed capsules or spheres to apply in Drug Dlievery System.Composite material constituted by spider silk protein and tissue engineering material become better histocompatibility and degradable material which will be used in the wide range of alternative tissue and sutures.
In some papers reported at present show that N-terminal non-repeat domain and C-terminal non-repeat domain are both very important in the process of silk self-assembly,The N-terminal domain comprises a secretion signal and is sensitive to pH. The C-terminal domainwas implicated in the control of solubility and fibre formation .Comparing to the N-terminal domain and the C-terminal domain, repeat domain have lower homology in different spider species and spider silks. Large molecular weight and complicated structure increase the research problem of repeat domain. Functions of repeat domain in the fiber formation process remain unassigned.We research the material property of silk fiber with different number of repeat domain in molecular biology and matcrialogy.
A 693bp partial fragment of spider flagelliform repeat domain was isolated from Araneus ventricosus gene library and this fragment contain an intact repeat domain of flagelliform silk gene. The gene was then optimized as E.coli codon and repeated 1-4 times respectively, meanwhile added the no-repeat N-terminal and C-terminal domains to the flank of the repeat fragments. Four recombination genes fused with intein and CBD (chitin binding domain) were expressed in IMPACT (Intein-Mediated Purification with an Affinity Chitin-binding Tag) system and purificated by affinity chromatography using chitin beads. DTT induced N-terminal cleavage of modified intein.Four target proteins N-Sc1/2/3/4-C could be elution from chitin beads without any exogenousamino acid residues.
Dialysis the pure proteins in pH 7.0 deion water respectively. The proteins after freeze drying are solubilized in 1, 1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 20%(wt/vol) for extrusion protein solutions into fibers by wet-spinning.Secondary structure in four kinds of biomimetic spider silks could be anlysised by Raman test. The structure of GPGGX in Repeat modules formβ-turn spiral which benefits the elasticity of spider silk. Protein secondary structure ofα-helix,β-sheet and random coil are mainly stored in N-terminal and C-terminal.The test results show there are not too many differences.The machenical propertiesof fibers with different repeat modulescould be checkouted by Stress-strain test. We can find flagilliform repeat domain mainly determine the extensibility of biomimetic spider silk.
All of my researches want to explain the number of spider flagelliform repeatdomains determine the fiber properties in the process of protein into fiber. We could find relationships between the amino acid sequences and the secondary structure of spider flagelliformrepeat domain. At the sametime, an efficiency system will be established to producegene engineering expressed biomimetic spider silk.
蜘蛛丝的基因主要由N端、C端非重复序列和中间的大量重复序列组成,目前对蜘蛛丝的研究主要分为：1,通过构建文库对不同种类蜘蛛不同类型蜘蛛丝全长基因的调取；2,研究蜘蛛丝蛋白成丝机理,主要涉及对N端、C端和中间序列在蜘蛛丝蛋白成丝过程中的作用,以及自组装过程中蛋白二级结构的变化；3,蜘蛛丝在生物医学工程方面的应用,主要涉及复合材料、药物缓释等内容。现在对蜘蛛丝的研究非常热,2010年连续在nature上发表了多篇关于蜘蛛丝成丝机理的研究论文。而且越来越多的人在关注蜘蛛丝在应用方面的研究,如Thomas R. Scheibel利用蜘蛛丝蛋白成丝的自组装来选择性包装和释放药物,可以在不同溶液中形成胶囊,可应用于药物缓释等方面。将蜘蛛丝蛋白溶液与现有的组织工程材料复合成新型的材料,期望得到更好组织相容性并可降解的材料,在替代性皮肤和手术缝合线方面会有很好的应用。
目前已经有很多文献报道蜘蛛丝基因表达的N端非重复模块可能构成蜘蛛丝的分泌信号并且是pH敏感元件,但是更多的功能还没被发现,C端非重复模块可以控制蜘蛛丝蛋白的可溶性和纤维形成,在丝纤维自组装过程中都起到重要的作用,但是由于不同种类蜘蛛和不同蛛丝之间重复模块的同源性没有N端和C端高,重复模块分子量大、结构复杂,增加了对其研究的难度。重复模块在纤维形成的过程中的功能还有待进一步研究,本课题从分子水平和材料学水平上对不同数量重复模块成丝性质进行了研究。
我们实验室在前期试验中,通过基因文库筛选得到一段大约693bp的大腹园蛛基因序列,经过分析和比对我们初步确认其为含有完整重复单元的鞭毛状丝基因。对这段基因根据原核生物大肠杆菌密码子使用频率进行基因优化后,依据鞭毛状丝的特点将这段基因分别拼接得到分别含有1-4段重复序列的质粒PE-Sc1/2/3/4,在重复片段的两端分别加上人工合成的蛛丝基因的N端和C端基因。将拼接得到的四段基因与IMPACT (Intein-Mediated Purification with an Affinity Chitin-binding Tag)系统的PTWIN1质粒融合表达得到N-Sc1/2/3/4-C-Intein-CBD蛋白(质粒PT-N-Sc1/2/3/4-C),利用几丁质结合蛋白与几丁质树脂的特异性结合亲和层析得到N-Sc1/2/3/4-C-Intein-CBD蛋白,通过加入DTT诱导Intein发生N端断裂,得到N-Scl/2/3/4-C蛋白
将纯化的蛋白透析至pH7.0的去离子水中,利用六氟异丙醇(HFIP)溶剂将冷冻干燥后的蛋白按照20%的质量体积分数回溶后,通过湿纺的手段得到四种单纤维类蜘蛛丝。组成丝纤维的四种丝蛋白单分子内N端和C端非重复结构的数量是一样的,不同的是逐渐增加的重复模块数量。通过对四种单丝的应力应变测试发现随着重复模块数增多,丝纤维的延展性越好,最好的是含有四段重复模块的丝纤维,应变程度可以达到25%。但是纤维的抗张强度并不是随着蛋白质分子量增加一直增强,含有四段重复模块的丝纤维应力为60MPa,而含有三段重复模块的丝纤维应力达到了120MPa,是含有四段重复模块丝纤维的两倍,说明重复模块对丝纤维强度并不是主要影响因素。
再对四种单丝进行显微激光Raman测试,分析仿生蜘蛛丝的内部二级结构组成,通过研究发现四种重组蛛丝中,都含有α-helix、β-sheet、β-turn和random coil蛋白质二级结构。N端和C端非重复模块不是鞭毛状丝的基因(N端、C端和重复模块氨基酸序列见附录),主要组成α-螺旋、β-折叠和无规则卷曲结构。四种丝纤维在蛋白质二级结构上,其α-螺旋、β-折叠和无规则卷曲结构含量是大体一致的。鞭毛状丝重复模块氨基酸序列主要都是由GPGGX的结构组成,形成β-turn二级结构,在此基础上形成β-turn螺旋,该结构能增强丝纤维的弹性,再次证明了鞭毛状丝重复模块的增加可以增强仿生蛛丝的延展性。
本课题主要从蛋白水平和材料学水平研究重复模块在成丝过程中的重要作用,期望能够为获得性能优越的仿生蛛丝的提供研究基础,同时也对基因工程仿生蛛丝的应用提供一种新的思路。
As a kind of protein material, spider silk has excellent material properties that can be used in aerospace, biomedical engineering, military projects such as flak suit. Although spider silk cannot be produced by simply raising spider like silkworm because of spider internecine nature instinct, scientists have tried to express spider silk gene in goat, plant, insect cell after the end of 20th century. However, the difficulties of overall length gene expression problem and low protein productivity have not been solved nowadays.
The gene of spider silk is mainly composed of N non-repeat terminal, C non-repeat terminal and many repeat modules between N-terminal and C-terminal. At present, the research of spider silk focus on aspects as followed:1, Constructing library to retrieve the overall length of spider silks from different species and glands; 2, Understanding spider silk protein into fiber mechanisms, mainly referring to the functions of N-terminal, C-terminal and middle repeat sequences in the process spider silk proteins transfer into fibers, also including proteins the change of proteins secondary structure in self-assembly. 3, Application of spider silk in biomedical engineering relate in composite material, drug delivery and so on. At the present, research of spider silk become more and more hot. Many research papers were continuously published in 2010 Nature magazine related the mechanism of spider silk protein transferring to protein fiber. More and more people pay close attention to application area of spider silk. Thomas R. Scheibel selectively embeded and released drug by self-assemble character of spider silk protein, formed capsules or spheres to apply in Drug Dlievery System.Composite material constituted by spider silk protein and tissue engineering material become better histocompatibility and degradable material which will be used in the wide range of alternative tissue and sutures.
In some papers reported at present show that N-terminal non-repeat domain and C-terminal non-repeat domain are both very important in the process of silk self-assembly,The N-terminal domain comprises a secretion signal and is sensitive to pH. The C-terminal domainwas implicated in the control of solubility and fibre formation .Comparing to the N-terminal domain and the C-terminal domain, repeat domain have lower homology in different spider species and spider silks. Large molecular weight and complicated structure increase the research problem of repeat domain. Functions of repeat domain in the fiber formation process remain unassigned.We research the material property of silk fiber with different number of repeat domain in molecular biology and matcrialogy.
A 693bp partial fragment of spider flagelliform repeat domain was isolated from Araneus ventricosus gene library and this fragment contain an intact repeat domain of flagelliform silk gene. The gene was then optimized as E.coli codon and repeated 1-4 times respectively, meanwhile added the no-repeat N-terminal and C-terminal domains to the flank of the repeat fragments. Four recombination genes fused with intein and CBD (chitin binding domain) were expressed in IMPACT (Intein-Mediated Purification with an Affinity Chitin-binding Tag) system and purificated by affinity chromatography using chitin beads. DTT induced N-terminal cleavage of modified intein.Four target proteins N-Sc1/2/3/4-C could be elution from chitin beads without any exogenousamino acid residues.
Dialysis the pure proteins in pH 7.0 deion water respectively. The proteins after freeze drying are solubilized in 1, 1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 20%(wt/vol) for extrusion protein solutions into fibers by wet-spinning.Secondary structure in four kinds of biomimetic spider silks could be anlysised by Raman test. The structure of GPGGX in Repeat modules formβ-turn spiral which benefits the elasticity of spider silk. Protein secondary structure ofα-helix,β-sheet and random coil are mainly stored in N-terminal and C-terminal.The test results show there are not too many differences.The machenical propertiesof fibers with different repeat modulescould be checkouted by Stress-strain test. We can find flagilliform repeat domain mainly determine the extensibility of biomimetic spider silk.
All of my researches want to explain the number of spider flagelliform repeatdomains determine the fiber properties in the process of protein into fiber. We could find relationships between the amino acid sequences and the secondary structure of spider flagelliformrepeat domain. At the sametime, an efficiency system will be established to producegene engineering expressed biomimetic spider silk.
引文
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[72]Wong Po Foo, C. et al. Nanocomposites from spider silksilica fusion (chimeric) proteins[J]. Proc. Natl. Acad. Sci. U. S. A,2006,103:9428-9433
[73]Huang, J. et al. The effect of genetically engineered spider silkdentin matrix protein 1 chimeric protein on hydroxyapatite nucleation[J].Biomaterials,2007,28:2358-2367
[74]A. Constans. Protein purification I:Liquid chromatography[J]. The Scientist,2002, 16(2):40.
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