CN114621352B - 硅融合蛋白及制备和应用 - Google Patents
硅融合蛋白及制备和应用 Download PDFInfo
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- CN114621352B CN114621352B CN202011456195.2A CN202011456195A CN114621352B CN 114621352 B CN114621352 B CN 114621352B CN 202011456195 A CN202011456195 A CN 202011456195A CN 114621352 B CN114621352 B CN 114621352B
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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Abstract
本发明公开了一种硅融合蛋白及制备和应用，主要涉及2，4‑二氧四氢蝶啶合成酶(Lumazine synthase，LS)与硅蛋白‑α(silicateinα)的融合蛋白及其纯化制备工艺。采用不同的融合方式获得四种融合蛋白基因，基因合成后插入pET30a载体构建重组质粒，转化大肠杆菌BL 21并筛选阳性克隆进行小规模培养及诱导表达。表达菌体经超声破碎、包涵体变复性处理、超滤浓缩及Superdex 200凝胶过滤层析纯化，最终获得电泳纯度大于95％的融合蛋白。硅催化活性检测结果显示，相对于未融合的硅蛋白α，本发明制备的四种硅融合蛋白，其催化活性均有显著的提高，本发明涉及的融合蛋白及其制备方法可为硅基材料及生物模板材料合成提供技术参考和物质基础。
Description
技术领域
本发明涉及生物硅化中硅蛋白研究领域，具体涉及一种高催化活性硅融合蛋白及其制备方法。
背景技术
硅元素在地壳中的丰度为28％，是仅次于氧元素丰度第二高的元素，广泛分布于生物圈中。其中一些生物能够用来构成稳定的结构，例如骨骼等。类似于这种生物矿化二氧化硅存在于多种生物中，很多生物(水稻，放射虫，领鞭虫、海绵等)中都有关于硅的生物矿化的报道。而生物机体内存在着多种生物活性的蛋白来协同完成整个生物矿化的过程。其中一种便是硅蛋白，其也可以在体外硅酸溶液中沉淀二氧化硅，这种活性来自其特有的蛋白序列和翻译后修饰调节。
2，4-二氧四氢蝶啶合成酶(Lumazine synthase，LS)，广泛存在于动植物及微生物中，为催化核黄素生物合成途径倒数第二步的酶。这种酶最显著的特征之一是在不同物种中具有空间结构差异，能够形成多聚体结构，形成对称性的衣壳。
研究显示，相对于传统的酸碱化学催化制备材料的方法，生物催化具有更高效、绿色、低能耗的特性；硅蛋白存在稳定性及催化活性低问题，且在以硅源为底物的催化反应中形成的二氧化硅以游离状态存在。针对这一问题，本发明选择将LS合成酶与Suberitesdomuncula(居蟹皮海绵)的硅蛋白silicateinα进行融合，使得硅蛋白在LS形成多聚体的空间三维结构衣壳表面展示，使得硅蛋白得到一个稳定富集的状态，从而提高硅蛋白的稳定性和活性。高催化活性的融合硅蛋白作为硅催化活性的原料提供一个更优的模板，对生物材料合成及探究具有一定的意义。
发明内容
本发明解决的技术问题是提供一种催化活性的融合硅蛋白的纯化制备方法，该方法与传统的化学催化方法相比，更高效、更环保。获得的融合硅蛋白，具有高表达、高活性、较为稳定的特性。
本发明为了解决上述技术问题采用如下技术方案：选择种属不同的LS合成酶，根据序列比对，选择高活性的来源于Aquifex aeolicus(嗜热菌)和Escherichia coli K-12(大肠杆菌)的LS合成酶，分别与硅蛋白α(根据序列比对，优选silicateinα-2序列，全文简称S2)的N端及C端通过不同方式融合表达，高活性的LS合成酶能够形成多聚体结构，例如二十面体对称性的衣壳，且在不同物种中存在空间结构的差异，选择不同物种来源的LS酶融合硅蛋白，将获取不同形貌的生物活性原材料。
N端或C端不同的融合表达方式，对于融合蛋白表达翻译及折叠产生不同的三维构象，将获得不同活性的融合蛋白。
S2蛋白在LS形成多聚体的空间三维结构衣壳展示富集，相对单一的硅蛋白，更加的稳定，且能够获得更高的表达量。
进一步地，融合蛋白经过包涵体变复性处理、超滤浓缩及Superdex 200凝胶过滤层析纯化，获得了较纯的蛋白，电泳纯度大于95％。
进一步地，通过纯化制备获得纯度较高的融合蛋白，使用钼酸比色法，TEOS作硅源，对纯化后的融合硅蛋白进行硅催化活性检测。实验数据表明，融合蛋白与阴性对照，即未融合的S2蛋白相比均具有较好的催化活性，尤其是S2-ALS融合蛋白，硅转化率可达约20％。
本发明与现有技术相比具有以下有益效果：相比于单一硅蛋白，活性低，不稳定，硅蛋白在催化体系中二氧化硅呈现大多数游离等问题，本发明通过LS融合硅蛋白表达，借助LS活性形成多面体衣壳，使得硅蛋白得到一个稳定富集的状态，达到高催化活性的目的，并且多面体结构可作为天然的模板进行材料合成，避免乳化剂及有机溶剂的使用。高催化活性的融合硅蛋白作为硅催化活性的原料提供一个优质的模板，对生物材料合成及探究具有一定的意义。
附图说明
图1为本发明一种高催化活性融合硅蛋白的不同融合表达方式示意图；
图2为本发明实施例3中S2-ELS融合硅蛋白包涵体变复性处理后还原性SDS-PAGE电泳分析图；
图3为本发明实施例3中ELS-S2融合硅蛋白包涵体变复性处理后还原性SDS-PAGE电泳分析图；
图4为本发明实施例3中ALS-S2融合硅蛋白包涵体变复性处理后还原性SDS-PAGE电泳分析图；
图5为本发明实施例3中S2-ALS融合硅蛋白包涵体变复性处理后还原性SDS-PAGE电泳分析图；
图6为本发明实施例3中S2-ELS融合硅蛋白凝胶过滤色谱图；
图7为本发明实施例3中ELS-S2融合硅蛋白凝胶过滤色谱图；
图8为本发明实施例3中ALS-S2融合硅蛋白凝胶过滤色谱图；
图9为本发明实施例3中S2-ALS融合硅蛋白凝胶过滤色谱图；
图10为本发明实施例3中S2-ELS、ELS-S2、ALS-S2、S2-ALS融合硅蛋白凝胶过滤层析纯化后还原性SDS-PAGE电泳分析图；
图11为本发明实施例3中S2-ELS、ELS-S2、ALS-S2、S2-ALS融合硅蛋白非还原性电泳图；
图12为本发明实施例4中纯化获得的S2-ELS、ELS-S2、ALS-S2、S2-ALS融合硅蛋白硅催化活性检测结果。
具体实施方式
为了能够更加详尽地了解本发明的特点与技术内容，下面对本发明的实现进行详细阐述。以下实施例中所描述的实验流程仅用于证明本发明的可行性，本发明的应用并不仅限于此。实施例中所提及的实验操作，如无特殊说明，均为常规实验方法；所提及的试剂耗材，如无特殊说明，均为常规试剂耗材。
实施例1融合蛋白重组质粒构建：
本发明一种高催化活性融合硅蛋白的制备方法，不同的融合表达方式示意图如图1所示，具体包括：选择来源于Aquifex aeolicus(嗜热菌)和Escherichia coli K-12(大肠杆菌)的LS合成酶，采用N端或C端的融合方式，委托金斯瑞公司合成融合蛋白基因，并插入pET30a(金斯瑞)载体构建重组质粒。
通过DNA合成公司，合成4种融合硅蛋白基因序列。
将合成的4种融合硅蛋白基因片段与原核表达载体pET30a连接，连接反应体系(20μl)如下：
共20μl体系，混匀，16℃连接过夜。经过酶切鉴定后，构建得到重组质粒，如图1所示。
实施例2阳性菌培养表达条件的优化：
将不同物种来源的LS合成酶与来源于Suberites domuncula的硅蛋白S2通过N端或C端不同的融合方式构建重组质粒，将重组质粒转入大肠杆菌BL21(DE3，CD601-02，全式金)，挑取单克隆尝试小量诱导表达尝试，确定较适的蛋白表达条件；
(1)取出一支50μL大肠杆菌感受态细胞BL21(DE3)于冰上放置，待解冻后加入2μL质粒(100ng/μL)，用手指轻轻弹拨混匀，冰上放置30min，42℃热击90s，冰上放置3min后加入350μL无抗性的的LB培养基，37℃，
180rpm孵育45min～1h，3000rpm离心2min，弃去250μL上清，轻轻吹打混匀，涂布在含有Kan抗性(100μg/mL，Solarbio，K8020-10g)的LB固体培养基上，37℃培养箱倒置过夜培养，获取单菌落。
(2)挑取2-4个转化后的单菌落分别接种至5mL含含有Kan抗性(100μg/mL)的LB液体培养基中，37℃，180rpm摇床过夜培养。保存菌种后1％的接菌量接种于含有Kan抗性(100μg/mL)的20mL LB培养基中，37℃、180rpm培养至OD600为0.4～0.6，分别进行18℃低温诱导和37℃诱导。
(3)18℃低温诱导：加入5μLIPTG(1mol/L，Solarbio，I8070-5g)诱导剂，18℃，70rpm诱导12h。
37℃诱导：加入5μL IPTG(1mol/L)诱导剂，37℃，180rpm诱导3h。(4)收集诱导后的菌体进行超声破碎，每一步收集样本进行SDS-PAGE电泳检测，分析不同温度诱导融合蛋白的表达情况。
实施例3蛋白表达及纯化：
在最适表达条件下对菌体进行扩大培养，诱导大量目标蛋白表达，获得的蛋白进行包涵体纯化、超滤浓缩及Superdex 200(GE 28990944)凝胶过滤层析纯化制备，最终获得较纯的蛋白，经SDS-PAGE电泳分析，纯度大于95％。
具体实验方法如下：
(1)蛋白表达：挑取重组质粒转化大肠杆菌BL21后涂布平板的单菌落于20mL含100μg/mL Kan的LB培养基中，37℃、180rpm培养过夜，按1％的比例接种于500mL含100μg/mLKan的LB培养基中，37℃、180rpm培养3h后加入IPTG至终浓度为0.25mmol/L进行诱导(125μL1M IPTG)，37℃、100rpm培养3h。4700rpm 4℃离心20min收集菌体。
(2)菌体破碎处理：取(1)中菌液各1管(30ml)加溶菌酶(Solarbio，L8120-50g终浓度1mg/mL)，冰上超声破碎(30％功率，破2s停2s，3min)，破碎2次。取破碎液80μL，加入20μL5×上样缓冲液，95℃变性10min，进行12％SDS-PAGE电泳分析。
(3)包涵体洗涤：破碎后12000rpm 4℃离心5min，取上清80μl，加入20μL
5×上样缓冲液，95℃变性10min，进行12％SDS-PAGE电泳分析。沉淀用1:1体积(30ml)的洗涤液(20mM Tris，2M UREA，50mM NaCl，0.5mM EDTA-2Na，0.1％Triton X-100，pH 8.0，试剂均来自Solarbio，为常用生物试剂)重悬洗涤，6000rpm 4℃离心3min重复3次，留取洗涤上清W1、W2、W3各取80μl，入20μL 5×上样缓冲液，95℃变性10min，进行12％
SDS-PAGE电泳分析。W4、W5各加10ml洗涤液洗涤。
(4)包涵体变性：洗涤后的沉淀用200μL复性液(20mM Tris，0.5mM EDTA，0.4mM氧化性GSH，0.3mM还原性GSH，10％甘油，0.02％Tween 80，2MUREA，pH 8.0)溶解，各加入3mL的变性液(20mM Tris，8M UREA，10mM DTT，pH 8.0)，室温放置2h后，12000rpm 4℃离心5min。取80μL，加入20μL 5×上样缓冲液，95℃变性10min，进行12％SDS-PAGE电泳分析。
(5)包涵体复性：烧杯中提前加入30倍体积的复性液，将变性液慢慢滴入复性液中，边滴边震荡。
(6)包涵体浓缩及缓冲液置换：对得到的样品用超滤离心管(UFC901096
millipore超滤离心管，截留分子量为10kDa)进行离心浓缩。采用3500rpm水平转子4℃离心20min，补加20mM Tris+50mM Nacl(pH 8.0)，继续离心，重复操作3～4次，最终50mL样本离心浓缩至5mL。取80μL，加入20μL 5×上样缓冲液，95℃变性10min，进行12％SDS-PAGE电泳分析。(7)凝胶过滤层析进一步纯化：选择Superdex 200(GE 28990944)预装柱，柱体积75mL，连接到AKTAPure蛋白纯化仪上，使用流动相A(20mM Tris+50mM NaCl pH8.0)平衡至响应值稳定，调零，保持基线平行；将透析浓缩后的样本通过系统泵过平衡好的柱子，设置为流速：1.0mL/min。根据紫外吸收响应值，收取主峰的馏分，12％SDS-PAGE电泳分析，根据电泳结果合并馏分，即得纯度合格的融合蛋白。以上结果如图2-11所示，所获得的融合蛋白经过多步的纯化，从电泳的胶图所示，目的条带较为明显，杂带与目的带相比，比较浅且模糊，目的蛋白的纯度大于95％。
实施例4对纯化的融合蛋白进行硅转化催化活性检测：
使用钼酸比色法，以TEOS(正硅酸乙酯)作硅源对纯化后的融合硅蛋白进行硅催化活性检测，结果如图12显示，四种硅融合蛋白的催化活性相较于未融合蛋白有显著的提高，其中S2-ALS的活性最高，达约20％。具体步骤如下：
取500μL蛋白于1.5mL Ep管中，再加入11μL预水解的TEOS(预水解条件：取3.3mLTEOS于15mL 1mM稀盐酸中水解30min)反应1h，反应结束后加入500μL无水乙醇混匀，12000rpm离心10min，最后用ddH2O洗涤3次。
利用硅钼黄测定法来定量硅。在1.5mL离心管中进行500μL二氧化硅沉淀反应。沉淀的二氧化硅用ddH2O洗涤3次，然后在室温下用40μL 2m NaOH溶解1小时。溶解后，加入160μL ddH2O和800μL硅钼酸盐溶液(1.35mL浓盐酸，40.3mL ddH2O，774.2mg四水合钼酸铵)并在室温下孵育15分钟，在室温下混合5分钟，静置10分钟。使用96孔板在405nm处测量吸光度。
使用以硅原子吸收标准品(Sigma#16259)进行的标准曲线将吸光度测量值转换为二氧化硅浓度。
根据以上实施例的方法与结果可知，本发明构建所获得的硅融合蛋白具有较好的催化活性，解决了单一的、未融合的硅蛋白不稳定、催化活性低，且在以硅源为底物的催化反应中形成的二氧化硅多数以游离状态存在等系列问题。相对于传统的酸碱化学催化制备材料的方法，本发明获得的硅融合蛋白可作为生物催化剂，具有更高效、绿色、低能耗的特性。并且硅融合蛋白亦可作为生物模板使用，提高反应效率，避免合成体系中有机溶剂的污染及安全风险，对生物材料合成探究及环境保护具有深远的意义。
以上实施例描述了本发明的基本设计原理、主要特征及优点，本行业的技术人员应该了解，本发明不受上述实施例的限制，上述实施例和说明书中描述的只是说明本发明的原理，只是用于帮助理解本发明，并不用以限制本发明。对于本发明所属技术领域的技术人员，依据本发明的思想，还可以做出若干简单推演、变形或替换。在不脱离本发明原理的范围下，本发明还会有各种变化和改进，这些变化和改进均落入本发明保护的范围内。
<110> 中国科学院大连化学物理研究所
<120> 硅融合蛋白及制备和应用
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Gly Cys His Gly Gly Asn Met Tyr Asp Ala Phe Leu Tyr Val Ile Ala
65 70 75 80
Asn Glu Gly Val Asp Gln Asp Ser Ala Tyr Pro Phe Val Gly Lys Gln
85 90 95
Ser Ser Cys Asn Tyr Asn Ser Lys Tyr Lys Gly Thr Ser Met Ser Gly
100 105 110
Met Val Ser Ile Lys Ser Gly Ser Glu Ser Asp Leu Gln Ala Ala Val
115 120 125
Ser Asn Val Gly Pro Val Ser Val Ala Ile Asp Gly Ala Asn Ser Ala
130 135 140
Phe Arg Phe Tyr Tyr Ser Gly Val Tyr Asp Gln Gln Gln Gln Gln Gln
145 150 155 160
Gln Gln Leu Asn His Ala Met Val Val Thr Gly Tyr Gly Ser Tyr Asn
165 170 175
Gly Lys Lys Tyr Trp Leu Ala Lys Asn Ser Trp Gly Thr Asn Trp Gly
180 185 190
Asn Ser Gly Tyr Val Met Met Ala Arg Asn Lys Tyr Asn Gln Cys Gly
195 200 205
Ile Ala Thr Asp Ala Ser Tyr Pro Thr Leu Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asn Ile Ile Glu Ala Asn Val
225 230 235 240
Ala Thr Pro Asp Ala Arg Val Ala Ile Thr Ile Ala Arg Phe Asn Asn
245 250 255
Phe Ile Asn Asp Ser Leu Leu Glu Gly Ala Ile Asp Ala Leu Lys Arg
260 265 270
Ile Gly Gln Val Lys Asp Glu Asn Ile Thr Val Val Trp Val Pro Gly
275 280 285
Ala Tyr Glu Leu Pro Leu Ala Ala Gly Ala Leu Ala Lys Thr Gly Lys
290 295 300
Tyr Asp Ala Val Ile Ala Leu Gly Thr Val Ile Arg Gly Gly Thr Ala
305 310 315 320
His Phe Glu Tyr Val Ala Gly Gly Ala Ser Asn Gly Leu Ala His Val
325 330 335
Ala Gln Asp Ser Glu Ile Pro Val Ala Phe Gly Val Leu Thr Thr Glu
340 345 350
Ser Ile Glu Gln Ala Ile Glu Arg Ala Gly Thr Lys Ala Gly Asn Lys
355 360 365
Gly Ala Glu Ala Ala Leu Thr Ala Leu Glu Met Ile Asn Val Leu Lys
370 375 380
Ala Ile Lys Ala
385
<210> 2
<211> 388
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Asn Ile Ile Glu Ala Asn Val Ala Thr Pro Asp Ala Arg Val Ala Ile
1 5 10 15
Thr Ile Ala Arg Phe Asn Asn Phe Ile Asn Asp Ser Leu Leu Glu Gly
20 25 30
Ala Ile Asp Ala Leu Lys Arg Ile Gly Gln Val Lys Asp Glu Asn Ile
35 40 45
Thr Val Val Trp Val Pro Gly Ala Tyr Glu Leu Pro Leu Ala Ala Gly
50 55 60
Ala Leu Ala Lys Thr Gly Lys Tyr Asp Ala Val Ile Ala Leu Gly Thr
65 70 75 80
Val Ile Arg Gly Gly Thr Ala His Phe Glu Tyr Val Ala Gly Gly Ala
85 90 95
Ser Asn Gly Leu Ala His Val Ala Gln Asp Ser Glu Ile Pro Val Ala
100 105 110
Phe Gly Val Leu Thr Thr Glu Ser Ile Glu Gln Ala Ile Glu Arg Ala
115 120 125
Gly Thr Lys Ala Gly Asn Lys Gly Ala Glu Ala Ala Leu Thr Ala Leu
130 135 140
Glu Met Ile Asn Val Leu Lys Ala Ile Lys Ala Gly Gly Gly Gly Ser
145 150 155 160
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Tyr Pro Glu Ala Val
165 170 175
Asp Trp Arg Thr Lys Gly Ala Val Thr Ala Val Lys Asp Gln Gly Asp
180 185 190
Cys Gly Ala Ser Tyr Ala Phe Ser Ala Met Gly Ala Leu Glu Gly Ala
195 200 205
Asn Ala Leu Ala Lys Gly Asn Ala Val Ser Leu Ser Glu Gln Asn Ile
210 215 220
Ile Asp Cys Ser Ile Pro Tyr Gly Asn His Gly Cys His Gly Gly Asn
225 230 235 240
Met Tyr Asp Ala Phe Leu Tyr Val Ile Ala Asn Glu Gly Val Asp Gln
245 250 255
Asp Ser Ala Tyr Pro Phe Val Gly Lys Gln Ser Ser Cys Asn Tyr Asn
260 265 270
Ser Lys Tyr Lys Gly Thr Ser Met Ser Gly Met Val Ser Ile Lys Ser
275 280 285
Gly Ser Glu Ser Asp Leu Gln Ala Ala Val Ser Asn Val Gly Pro Val
290 295 300
Ser Val Ala Ile Asp Gly Ala Asn Ser Ala Phe Arg Phe Tyr Tyr Ser
305 310 315 320
Gly Val Tyr Asp Gln Gln Gln Gln Gln Gln Gln Gln Leu Asn His Ala
325 330 335
Met Val Val Thr Gly Tyr Gly Ser Tyr Asn Gly Lys Lys Tyr Trp Leu
340 345 350
Ala Lys Asn Ser Trp Gly Thr Asn Trp Gly Asn Ser Gly Tyr Val Met
355 360 365
Met Ala Arg Asn Lys Tyr Asn Gln Cys Gly Ile Ala Thr Asp Ala Ser
370 375 380
Tyr Pro Thr Leu
385
<210> 3
<211> 386
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Gln Ile Tyr Glu Gly Lys Leu Thr Ala Glu Gly Leu Arg Phe Gly Ile
1 5 10 15
Val Ala Ser Arg Phe Asn His Ala Leu Val Asp Arg Leu Val Glu Gly
20 25 30
Ala Ile Asp Cys Ile Val Arg His Gly Gly Arg Glu Glu Asp Ile Thr
35 40 45
Leu Val Arg Val Pro Gly Ser Trp Glu Ile Pro Val Ala Ala Gly Glu
50 55 60
Leu Ala Arg Lys Glu Asp Ile Asp Ala Val Ile Ala Ile Gly Val Leu
65 70 75 80
Ile Arg Gly Ala Thr Pro His Phe Asp Tyr Ile Ala Ser Glu Val Ser
85 90 95
Lys Gly Leu Ala Asn Leu Ser Leu Glu Leu Arg Lys Pro Ile Thr Phe
100 105 110
Gly Val Ile Thr Ala Asp Thr Leu Glu Gln Ala Ile Glu Arg Ala Gly
115 120 125
Thr Lys His Gly Asn Lys Gly Trp Glu Ala Ala Leu Ser Ala Ile Glu
130 135 140
Met Ala Asn Leu Phe Lys Ser Leu Arg Gly Gly Gly Gly Ser Gly Gly
145 150 155 160
Gly Gly Ser Gly Gly Gly Gly Ser Asp Tyr Pro Glu Ala Val Asp Trp
165 170 175
Arg Thr Lys Gly Ala Val Thr Ala Val Lys Asp Gln Gly Asp Cys Gly
180 185 190
Ala Ser Tyr Ala Phe Ser Ala Met Gly Ala Leu Glu Gly Ala Asn Ala
195 200 205
Leu Ala Lys Gly Asn Ala Val Ser Leu Ser Glu Gln Asn Ile Ile Asp
210 215 220
Cys Ser Ile Pro Tyr Gly Asn His Gly Cys His Gly Gly Asn Met Tyr
225 230 235 240
Asp Ala Phe Leu Tyr Val Ile Ala Asn Glu Gly Val Asp Gln Asp Ser
245 250 255
Ala Tyr Pro Phe Val Gly Lys Gln Ser Ser Cys Asn Tyr Asn Ser Lys
260 265 270
Tyr Lys Gly Thr Ser Met Ser Gly Met Val Ser Ile Lys Ser Gly Ser
275 280 285
Glu Ser Asp Leu Gln Ala Ala Val Ser Asn Val Gly Pro Val Ser Val
290 295 300
Ala Ile Asp Gly Ala Asn Ser Ala Phe Arg Phe Tyr Tyr Ser Gly Val
305 310 315 320
Tyr Asp Gln Gln Gln Gln Gln Gln Gln Gln Leu Asn His Ala Met Val
325 330 335
Val Thr Gly Tyr Gly Ser Tyr Asn Gly Lys Lys Tyr Trp Leu Ala Lys
340 345 350
Asn Ser Trp Gly Thr Asn Trp Gly Asn Ser Gly Tyr Val Met Met Ala
355 360 365
Arg Asn Lys Tyr Asn Gln Cys Gly Ile Ala Thr Asp Ala Ser Tyr Pro
370 375 380
Thr Leu
385
<210> 4
<211> 387
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Asp Tyr Pro Glu Ala Val Asp Trp Arg Thr Lys Gly Ala Val Thr Ala
1 5 10 15
Val Lys Asp Gln Gly Asp Cys Gly Ala Ser Tyr Ala Phe Ser Ala Met
20 25 30
Gly Ala Leu Glu Gly Ala Asn Ala Leu Ala Lys Gly Asn Ala Val Ser
35 40 45
Leu Ser Glu Gln Asn Ile Ile Asp Cys Ser Ile Pro Tyr Gly Asn His
50 55 60
Gly Cys His Gly Gly Asn Met Tyr Asp Ala Phe Leu Tyr Val Ile Ala
65 70 75 80
Asn Glu Gly Val Asp Gln Asp Ser Ala Tyr Pro Phe Val Gly Lys Gln
85 90 95
Ser Ser Cys Asn Tyr Asn Ser Lys Tyr Lys Gly Thr Ser Met Ser Gly
100 105 110
Met Val Ser Ile Lys Ser Gly Ser Glu Ser Asp Leu Gln Ala Ala Val
115 120 125
Ser Asn Val Gly Pro Val Ser Val Ala Ile Asp Gly Ala Asn Ser Ala
130 135 140
Phe Arg Phe Tyr Tyr Ser Gly Val Tyr Asp Gln Gln Gln Gln Gln Gln
145 150 155 160
Gln Gln Leu Asn His Ala Met Val Val Thr Gly Tyr Gly Ser Tyr Asn
165 170 175
Gly Lys Lys Tyr Trp Leu Ala Lys Asn Ser Trp Gly Thr Asn Trp Gly
180 185 190
Asn Ser Gly Tyr Val Met Met Ala Arg Asn Lys Tyr Asn Gln Cys Gly
195 200 205
Ile Ala Thr Asp Ala Ser Tyr Pro Thr Leu Gly Gly Gly Gly Ser Gly
210 215 220
Gly Gly Gly Ser Gly Gly Gly Gly Ser Met Gln Ile Tyr Glu Gly Lys
225 230 235 240
Leu Thr Ala Glu Gly Leu Arg Phe Gly Ile Val Ala Ser Arg Phe Asn
245 250 255
His Ala Leu Val Asp Arg Leu Val Glu Gly Ala Ile Asp Cys Ile Val
260 265 270
Arg His Gly Gly Arg Glu Glu Asp Ile Thr Leu Val Arg Val Pro Gly
275 280 285
Ser Trp Glu Ile Pro Val Ala Ala Gly Glu Leu Ala Arg Lys Glu Asp
290 295 300
Ile Asp Ala Val Ile Ala Ile Gly Val Leu Ile Arg Gly Ala Thr Pro
305 310 315 320
His Phe Asp Tyr Ile Ala Ser Glu Val Ser Lys Gly Leu Ala Asn Leu
325 330 335
Ser Leu Glu Leu Arg Lys Pro Ile Thr Phe Gly Val Ile Thr Ala Asp
340 345 350
Thr Leu Glu Gln Ala Ile Glu Arg Ala Gly Thr Lys His Gly Asn Lys
355 360 365
Gly Trp Glu Ala Ala Leu Ser Ala Ile Glu Met Ala Asn Leu Phe Lys
370 375 380
Ser Leu Arg
385
<210> 5
<211> 1179
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ggatccgact acccggaagc ggttgattgg cgtaccaagg gcgcggtgac cgcggttaaa 60
gaccagggtg attgcggcgc gagctatgcg ttcagcgcga tgggtgcgct ggagggtgcg 120
aacgcgctgg cgaagggtaa cgcggtgagc ctgagcgaac aaaacatcat tgactgcagc 180
atcccgtacg gtaaccacgg ctgccacggt ggcaacatgt acgatgcgtt cctgtatgtg 240
attgcgaacg aaggtgttga ccaggatagc gcgtatccgt ttgtgggcaa gcaaagcagc 300
tgcaactaca acagcaagta taaaggtacc agcatgagcg gcatggttag catcaaaagc 360
ggtagcgaga gcgacctgca ggcggcggtg agcaacgttg gtccggtgag cgttgcgatt 420
gacggcgcga acagcgcgtt ccgtttttac tatagcggtg tgtacgatca gcaacaacag 480
cagcaacagc aactgaacca cgcgatggtg gttaccggtt acggcagcta taacggtaag 540
aaatattggc tggcgaagaa cagctggggc accaactggg gtaacagcgg ctacgttatg 600
atggcgcgta acaaatataa ccaatgcggt atcgcgaccg atgcgagcta cccgaccctg 660
ggtggcggtg gcagcggtgg cggtggcagc ggtggcggtg gcagcaacat cattgaagcg 720
aacgttgcga ccccggatgc gcgtgttgcg atcaccattg cgcgtttcaa caactttatc 780
aacgacagcc tgctggaggg tgcgatcgat gcgctgaagc gtattggcca ggtgaaagac 840
gagaacatta ccgtggtttg ggttccgggt gcgtatgaac tgccgctggc ggcgggtgcg 900
ctggcgaaga ccggcaaata tgatgcggtg atcgcgctgg gtaccgttat tcgtggtggc 960
accgcgcact ttgagtatgt ggcgggtggc gcgagcaacg gtctggcgca tgttgcgcag 1020
gatagcgaaa tcccggtggc gtttggcgtt ctgaccaccg agagcatcga acaagcgatt 1080
gagcgtgcgg gtaccaaagc gggtaacaag ggtgcggagg cggcgctgac cgcgctggaa 1140
atgatcaacg tgctgaaggc gattaaagcg taactcgag 1179
<210> 6
<211> 1179
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ggatccaaca tcattgaagc gaacgttgcg accccggatg cgcgtgttgc gatcaccatt 60
gcgcgtttca acaactttat caacgacagc ctgctggagg gtgcgatcga tgcgctgaag 120
cgtattggcc aggtgaaaga cgagaacatt accgtggttt gggttccggg tgcgtatgaa 180
ctgccgctgg cggcgggtgc gctggcgaag accggcaaat atgatgcggt gatcgcgctg 240
ggtaccgtta ttcgtggtgg caccgcgcac tttgagtatg tggcgggtgg cgcgagcaac 300
ggtctggcgc atgttgcgca ggatagcgaa atcccggtgg cgtttggcgt tctgaccacc 360
gagagcatcg aacaagcgat tgagcgtgcg ggtaccaaag cgggtaacaa gggtgcggag 420
gcggcgctga ccgcgctgga aatgatcaac gtgctgaagg cgattaaagc gggtggcggt 480
ggcagcggtg gcggtggcag cggtggcggt ggcagcgact acccggaagc ggttgattgg 540
cgtaccaagg gcgcggtgac cgcggttaaa gaccagggtg attgcggcgc gagctatgcg 600
ttcagcgcga tgggtgcgct ggagggtgcg aacgcgctgg cgaagggtaa cgcggtgagc 660
ctgagcgaac aaaacatcat tgactgcagc atcccgtacg gtaaccacgg ctgccacggt 720
ggcaacatgt acgatgcgtt cctgtatgtg attgcgaacg aaggtgttga ccaggatagc 780
gcgtatccgt ttgtgggcaa gcaaagcagc tgcaactaca acagcaagta taaaggtacc 840
agcatgagcg gcatggttag catcaaaagc ggtagcgaga gcgacctgca ggcggcggtg 900
agcaacgttg gtccggtgag cgttgcgatt gacggcgcga acagcgcgtt ccgtttttac 960
tatagcggtg tgtacgatca gcaacaacag cagcaacagc aactgaacca cgcgatggtg 1020
gttaccggtt acggcagcta taacggtaag aaatattggc tggcgaagaa cagctggggc 1080
accaactggg gtaacagcgg ctacgttatg atggcgcgta acaaatataa ccaatgcggt 1140
atcgcgaccg atgcgagcta cccgaccctg taactcgag 1179
<210> 7
<211> 1173
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ggatcccaga tttacgaggg caagctgacc gcggaaggtc tgcgtttcgg catcgtggcg 60
agccgtttta accacgcgct ggttgaccgt ctggttgagg gtgcgatcga ttgcattgtt 120
cgtcacggtg gccgtgagga agacattacc ctggtgcgtg ttccgggtag ctgggagatt 180
ccggtggcgg cgggtgaact ggcgcgtaaa gaagacatcg atgcggtgat cgcgattggt 240
gttctgattc gtggcgcgac cccgcacttc gactatatcg cgagcgaggt gagcaagggt 300
ctggcgaacc tgagcctgga actgcgtaaa ccgatcacct ttggcgttat taccgcggat 360
accctggagc aggcgattga acgtgcgggt accaagcacg gtaacaaggg ttgggaggcg 420
gcgctgagcg cgatcgaaat ggcgaacctg ttcaaaagcc tgcgtggtgg cggtggcagc 480
ggtggcggtg gcagcggtgg cggtggcagc gactacccgg aggcggtgga ttggcgtacc 540
aagggcgcgg tgaccgcggt taaagaccag ggtgattgcg gcgcgagcta tgcgtttagc 600
gcgatgggtg cgctggaggg tgcgaacgcg ctggcgaagg gtaacgcggt tagcctgagc 660
gaacaaaaca tcattgactg cagcattccg tacggtaacc acggctgcca cggtggcaac 720
atgtacgatg cgttcctgta tgtgatcgcg aacgagggtg ttgaccagga tagcgcgtat 780
ccgtttgtgg gcaagcaaag cagctgcaac tacaacagca agtataaagg taccagcatg 840
agcggcatgg ttagcattaa aagcggtagc gaaagcgacc tgcaggcggc ggtgagcaac 900
gttggtccgg tgagcgttgc gattgatggt gcgaacagcg cgttccgttt ttactatagc 960
ggtgtgtacg atcagcaaca acagcagcaa cagcaactga accacgcgat ggtggttacc 1020
ggttacggca gctataacgg taagaaatat tggctggcga agaacagctg gggcaccaac 1080
tggggtaaca gcggctacgt tatgatggcg cgtaacaaat ataaccaatg cggtatcgcg 1140
accgatgcga gctacccgac cctgtaactc gag 1173
<210> 8
<211> 1176
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
ggatccgact acccggaggc ggtggattgg cgtaccaagg gcgcggtgac cgcggttaaa 60
gaccagggtg attgcggcgc gagctatgcg ttcagcgcga tgggtgcgct ggagggtgcg 120
aacgcgctgg cgaagggtaa cgcggttagc ctgagcgaac aaaacatcat tgactgcagc 180
attccgtacg gtaaccacgg ctgccacggt ggcaacatgt acgatgcgtt cctgtatgtg 240
atcgcgaacg agggtgttga ccaggatagc gcgtatccgt ttgtgggcaa gcaaagcagc 300
tgcaactaca acagcaagta taaaggtacc agcatgagcg gcatggttag cattaaaagc 360
ggtagcgaaa gcgacctgca ggcggcggtg agcaacgttg gtccggtgag cgttgcgatt 420
gatggtgcga acagcgcgtt ccgtttttac tatagcggtg tgtacgatca gcaacaacag 480
cagcaacagc aactgaacca cgcgatggtg gttaccggtt acggcagcta taacggtaag 540
aaatattggc tggcgaagaa cagctggggc accaactggg gtaacagcgg ctacgttatg 600
atggcgcgta acaaatataa ccagtgcggt atcgcgaccg acgcgagcta tccgaccctg 660
ggtggcggtg gcagcggtgg cggtggcagc ggtggcggtg gcagcatgca aatttatgag 720
ggcaagctga ccgcggaagg tctgcgtttc ggcatcgtgg cgagccgttt taaccacgcg 780
ctggttgacc gtctggttga gggtgcgatc gattgcattg ttcgtcacgg tggccgtgag 840
gaagatatta ccctggtgcg tgttccgggt agctgggaga ttccggtggc ggcgggtgaa 900
ctggcgcgta aagaagacat cgatgcggtg atcgcgattg gtgttctgat tcgtggcgcg 960
accccgcact tcgactacat cgcgagcgag gtgagcaagg gtctggcgaa cctgagcctg 1020
gaactgcgta aaccgatcac ctttggcgtt attaccgcgg ataccctgga gcaggcgatt 1080
gaacgtgcgg gtaccaagca cggtaacaag ggttgggagg cggcgctgag cgcgatcgaa 1140
atggcgaacc tgtttaaaag cctgcgttaa ctcgag 1176
Claims (5)
1.硅融合蛋白，其特征在于，所述的硅融合蛋白包括LS合成酶和硅蛋白α以不同方式连接的硅融合蛋白，硅融合蛋白的氨基酸序列如SEQ ID NO：1、2和4所示中的一种。
2. 一种编码硅融合蛋白的多核苷酸，其特征在于，所述多核苷酸的序列如SEQ ID NO：5、6和8所示中的一种。
3. 一种硅融合蛋白的纯化制备方法，构建含有编码权利要求1所述的硅融合蛋白目的基因的表达载体，导入大肠杆菌宿主细胞中表达，表达菌体通过破碎、包涵体变复性处理、超滤浓缩及Superdex 200凝胶过滤层析纯化单元操作制备过程，最终获得电泳纯度大于95%的高催化活性的目标蛋白。
4.如权利要求1所述的融合蛋白在硅基材料合成中的应用。
5.根据权利要求4中所述的应用，其特征在于，将融合蛋白作为合成反应的催化剂或生物模板使用。
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| CN111217918A (zh) * | 2020-03-04 | 2020-06-02 | 中山大学 | 一种基于2,4-二氧四氢喋啶合酶的新型冠状病毒s蛋白双区域亚单位纳米疫苗 |
| CN114588874A (zh) * | 2020-12-04 | 2022-06-07 | 中国科学院大连化学物理研究所 | 一种基于海绵硅蛋白的多孔材料及制备和应用 |
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| AR044603A1 (es) * | 2004-06-03 | 2005-09-21 | Consejo Nac Invest Cient Tec | Proteinas quimericas aisladas de lumazina sintetasa modificada para la presentacion multiple de moleculas y sus aplicaciones |
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| CN111217918A (zh) * | 2020-03-04 | 2020-06-02 | 中山大学 | 一种基于2,4-二氧四氢喋啶合酶的新型冠状病毒s蛋白双区域亚单位纳米疫苗 |
| CN114588874A (zh) * | 2020-12-04 | 2022-06-07 | 中国科学院大连化学物理研究所 | 一种基于海绵硅蛋白的多孔材料及制备和应用 |
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