CN102660524B - 高表达高比活皮蝇胶原酶突变体序列、毕赤酵母表达质粒的构建及菌株筛选和纯化方法 - Google Patents
高表达高比活皮蝇胶原酶突变体序列、毕赤酵母表达质粒的构建及菌株筛选和纯化方法 Download PDFInfo
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Abstract
本发明涉及皮蝇胶原酶（HlCollagenase）突变体的序列及其分泌表达的酵母菌株HlCollagenase-m/pGAPZα-A/GS115构建的关键技术、构建结果及利用它来生产高活性皮蝇胶原酶的纯化方法。本发明采用基因工程表达的方法生产高活性的皮蝇胶原酶突变体，比活性达到1320国际单位/毫克,表达量达到241毫克/升蛋白，通过纯化可获得纯度达95%以上的目标蛋白皮蝇胶原酶，为皮蝇胶原酶今后的推广使用奠定良好基础。
Description
技术领域
本发明涉及具有高比活皮蝇胶原酶（Hl Collagenase） 序列的确认、该序列毕赤酵母表达载体的构建、分泌表达皮蝇胶原酶的毕赤酵母菌株Hl Collagenase-m/ pGAPZα-A/GS115构建的关键技术、构建结果及利用它来生产高活性皮蝇胶原酶的纯化方法。本发明属于生物技术领域。
背景技术
胶原酶的化学名为胶原蛋白水解酶(Collagenase)，它能在生理PH和温度条件下特异性地水解天然胶原蛋白的三维螺旋结构，而不损伤其它蛋白质和组织。来自梭菌的胶原酶，切割胶原蛋白中甘氨酸残基的氨基端肽键。而来自皮蝇(Hypoderma lineatum)幼虫的胶原酶，切割天然胶原蛋白丙氨酸残基的氨基端肽键。目前医疗上广泛使用的胶原酶是从溶组织梭状细胞芽孢杆菌提取制备的，主要水解***中胶原蛋白成分。而来自皮蝇幼虫的胶原酶，由于幼虫体内含量低，提取工艺复杂，生产成本高，至今无法大量生产，阻碍了皮蝇胶原酶的推广使用。
发明内容
皮蝇胶原酶是皮蝇一期幼虫可溶性抗原的主要成分，是一种丝氨酸蛋白酶，分子量为25223道尔顿，成熟的皮蝇胶原酶含有230个氨基酸，比其前体少了30个氨基酸，整个氨基酸序列和胰蛋白酶家族的丝氨酸蛋白酶的氨基酸序列具有高度的相似性。其活性功能片段为45~180位氨基酸,底物结合区是174~201位氨基酸；全序列共有6个半胱氨酸（30,46；151,166；176,204位氨基酸），形成3对二硫键。根据对皮蝇胶原酶分子结构及序列的分析，我们设计切除1~27位的氨基酸序列，使功能区域更大范围地暴露，使之能够更好的和反应底物结合，从而提高酶的活性。并将第29位的色氨酸（Trp）突变为异亮氨酸(Ile),以保持分子结构的稳定性；
具有高活性的皮蝇胶原酶突变体序列如下：
Val Ile CysGlyGlySerLeuIleAspAsnLysTrpIleLeuThrAlaAlaHisCysValHisAspAlaValSerValValValTyrLeuGlySerAlaValGlnTyrGluGlyGluAlaValValAsnSerGluArgIleIleSerHisSerMetPheAsnProAspThrTyrLeuAsnAspValAlaLeuIleLysIleProHisValGluTyrThrAspAsnIleGlnProIleArgLeuProSerGlyGluGluLeuAsnAsnLysPheGluAsnIleTrpAlaThrValSerGlyTrpGlyGlnSerAsnThrAspThrValIleLeuGlnTyrThrTyrAsnLeuValIleAspAsnAspArgCysAlaGlnGluTyrProProGlyIleIleValGluSerThrIleCysGlyAspThrSerAspGlyLysSerProCysPheGlyAspSerGlyGlyProPheValLeuSerAspLysAsnLeuLeuIleGlyValValSerPheValSerGlyAlaGlyCysGluSerGlyLysProValGlyPheSerArgValThrSerTyrMetAspTrpIleGlnGlnAsnThrGlyIleLysPhe
该序列的特点是只保留了成熟皮蝇胶原酶序列中的功能区域和底物结合区（28~230位氨基酸）并将29位的色氨酸点突变为亮氨酸，这种改变在理论上使功能区域更大范围地暴露，使之能够更好的和反应底物结合，从而提高酶的活性；
同时，本发明采用毕赤酵母分泌表达***，该***产量高、生产成本低兼有真核表达***翻译后修饰功能等优点，可以用于大量生产皮蝇胶原酶，降低生产成本；
皮蝇胶原酶毕赤酵母重组菌株的构建方法为：将修饰突变的皮蝇胶原酶Hl Collagenase基因***pGAPZα-A空载体的GAP启动子/α-factor信号肽下游位点，并删除载体上的Kex2位点Lys-Arg之后的Ste13位点Glu-Ala-Glu-Ala,构建成皮蝇胶原酶基因分泌型表达载体Hl Collagenase-m/ pGAPZα-A；利用电转化的方法将Collagenase-m/ pGAPZα-A载体转化毕赤酵母（Pichia pastoris）的菌株GS115，构建成分泌型表达皮蝇胶原酶突变体的毕赤酵母工程菌株--- Hl Collagenase-m/ pGAPZα-A/GS115；通过抗生素筛选、SDS-PAGE电泳分析及生物比活性测定，获得高表达高活性的皮蝇胶原酶突变体毕赤酵母重组菌株；
上述的毕赤酵母重组菌株目标蛋白：皮蝇胶原酶突变体（Hl Collagenase-m）的表达量达到241毫克/升，比活性为1320国际单位/毫克；目标蛋白皮蝇胶原酶突变体比天然的皮蝇胶原酶比活性高5~6倍，且具有很高的稳定性；
皮蝇胶原酶突变体的纯化方法为：利用获得高表达高活性的皮蝇胶原酶突变体毕赤酵母重组菌株进行发酵，离心收集上清液，以氢氧化钠调节PH值，过阴离子交换层析柱，收集目标洗脱峰，得到纯度约70%的皮蝇胶原酶突变体粗品，透析除盐，超滤浓缩，过分子筛层析柱，收集目标蛋白，得到纯度大于95%的皮蝇胶原酶突变体蛋白。
【附图说明】
附图为皮蝇胶原酶突变体（Hl Collagenase-m）表达载体构建图
Hl Collagenase-m/ pGAPZα-A表达载体分子大小为3.759kb。其中pGAPZα-A为3.147kb，Hl Collagenase-m为612bp(包含终止子密码)；Hl Collagenase-m***于载体pGAPZα-A第747与824bp（XbaI位点）之间；
pGAPZα-A载体结构为：
磷酸甘油酸脱氢酶基因（GAP）启动子区域：第1-483bp
磷酸甘油酸脱氢酶基因启动子引物位点：第455-476bp
α-交配因子分泌信号肽序列：第493-759bp
α-交配因子分泌信号肽引物位点：第696-716bp
载体多克隆位点：第760-828bp
Myc抗原决定簇包：第827-856bp
3’-乙醇氧化酶1基因引物位点：第974-994bp
乙醇氧化酶1转录终止区域：第593-1233bp
转录延伸因子1启动子区域：第1234-1644bp
合成原核启动子：第1645-1712bp
链霉菌zeocin抗性基因阅读框：第1713-2087bp
细胞色素合成酶1转录终止区域：第2088-2405bp
大肠杆菌复制子1（来源于pUC载体）：第2416-3089bp。
【具体实施方式】
下面结合实施例详细介绍本发明在皮蝇胶原酶突变体重组毕赤酵母工程菌株的构建和皮蝇胶原酶突变体纯化工艺中的具体应用；
实施例1
皮蝇胶原酶突变体（Hl Collagenase-m）毕赤酵母工程菌的构建
1、 根据皮蝇胶原酶突变体的氨基酸序列和酵母对密码子的偏好，设计皮蝇胶原酶突变体全核苷酸序列，并在序列两端引入相应的酶切位点XhoI和XbaI，由大连宝生物公司合成该序列；
2、 采用基因克隆技术将皮蝇胶原酶突变体基因经XhoI/XbaI双酶切后***pGAPZα-A之GAP启动子/α-factor信号肽下游的XhoI/XbaI位点，构建成含α-factor信号肽的分泌型毕赤酵母表达载体；Hl Collagenase-m/ pGAPZα-A表达载体分子大小为3.759kb,其中pGAPZα-A为3.147kb，Hl Collagenase-m为612bp(包含终止子)，皮蝇胶原酶突变体***于载体pGAPZα-A第747bp与824bp(XbaI位点)之间，具体情况见附图；
3、 通过电转化方法，将线性化的Hl Collagenase-m/ pGAPZα-A质粒转入毕赤酵母GS115感受态（购于Invitrogen公司）中，通过zeocin抗生素筛选获得阳性菌株，进一步用PCR和Southern blotting技术鉴定筛选出的阳性菌株，再用SDS-PAGE电泳方法和SDS-PAGE活性测定方法筛选高表达高活性的Hl Collagenase-m/ pGAPZα-A/GS115工程菌株；
实施例2
皮蝇胶原酶突变体的纯化方法
1、 从-80℃种子库中取出Hl Collagenase-m/ pGAPZα-A/GS115工程菌，接种平板，使菌株活化；
2、 从平板中挑单菌落于200毫升YPD+zeocin100毫克/升培养基中，30℃,250转/分钟，培养48小时，此为一级种子液；
3、 吸取100毫升的一级种子液接种于2升的YPD培养基中，30℃,250转/分钟，培养24小时，此为二级种子液；
4、 将2升的二级种子液接种于含40升YPD培养基的发酵罐中，发酵72小时，离心收集发酵上清液，以1摩尔氢氧化钠溶液调节PH8.0-9.0，过DEAE SepharoseFF层析柱，收集0.3摩尔/升氯化钠溶液洗脱峰，透析过夜除盐，此时可得到纯度大约为70%左右的皮蝇胶原酶突变体蛋白粗品，超滤浓缩，过G25层析柱，收集目标蛋白峰，得到纯度大于95%的皮蝇胶原酶突变体蛋白原液，将原液冻干保存。
<110> 海南华研生物科技有限公司
<120>高表达高比活皮蝇胶原酶突变体序列、毕赤酵母表达质粒的构建及菌株筛选和纯化方法
<160> 1
<170> PatentIn Version 2.1
<210> 1
<211> 203
<212>氨基酸
<213> Hypoderma lineatum (early cattle grub)
<220>
<221> misc_difference
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Ala His Cys Val His Asp Ala Val Ser Val Val Val Tyr Leu Gly Ser
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Ala Val Gln Tyr Glu Gly Glu Ala Val Val Asn Ser Glu Arg Ile Ile
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Ser His Ser Met Phe Asn Pro Asp Thr Tyr Leu Asn Asp Val Ala Leu
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Ile Lys Ile Pro His Val Glu Tyr Thr Asp Asn Ile Gln Pro Ile Arg
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Leu Pro Ser Gly Glu Glu Leu Asn Asn Lys Phe Glu Asn Ile Trp Ala
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Thr Val Ser Gly Trp Gly Gln Ser Asn Thr Asp Thr Val Ile Leu Gln
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Tyr Thr Tyr Asn Leu Val Ile Asp Asn Asp Arg Cys Ala Gln Glu Tyr
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Pro Pro Gly Ile Ile Val Glu Ser Thr Ile Cys Gly Asp Thr Ser Asp
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Asp Lys Asn Leu Leu Ile Gly Val Val Ser Phe Val Ser Gly Ala Gly
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Cys Glu Ser Gly Lys Pro Val Gly Phe Ser Arg Val Thr Ser Tyr Met
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Asp Trp Ile Gln Gln Asn Thr Gly Ile Lys Phe
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Claims (8)
1.一种皮蝇胶原酶（Hl Collagenase）突变体，其氨基酸序列为如下序列
Val Ile CysGlyGlySerLeuIleAspAsnLysTrpIleLeuThrAlaAlaHisCysValHisAspAlaValSerValValValTyrLeuGlySerAlaValGlnTyrGluGlyGluAlaValValAsnSerGluArgIleIleSerHisSerMetPheAsnProAspThrTyrLeuAsnAspValAlaLeuIleLysIleProHisValGluTyrThrAspAsnIleGlnProIleArgLeuProSerGlyGluGluLeuAsnAsnLysPheGluAsnIleTrpAlaThrValSerGlyTrpGlyGlnSerAsnThrAspThrValIleLeuGlnTyrThrTyrAsnLeuValIleAspAsnAspArgCysAlaGlnGluTyrProProGlyIleIleValGluSerThrIleCysGlyAspThrSerAspGlyLysSerProCysPheGlyAspSerGlyGlyProPheValLeuSerAspLysAsnLeuLeuIleGlyValValSerPheValSerGlyAlaGlyCysGluSerGlyLysProValGlyPheSerArgValThrSerTyrMetAspTrpIleGlnGlnAsnThrGlyIleLysPhe。
2.一种皮蝇胶原酶突变体表达载体，其特征在于表达质粒中转入了权利要求1所述的皮蝇胶原酶突变体的编码基因。
3.根据权利要求2所述的皮蝇胶原酶突变体表达载体，其特征在于所述的表达质粒为pGAPZα-A。
4.一种皮蝇胶原酶突变体重组表达载体，其特征在于是将权利要求1所述的皮蝇胶原酶Hl Collagenase突变体的编码基因***pGAPZα-A空载体的GAP启动子/α-factor信号肽下游位点，并删除载体上的Kex2位点Lys-Arg之后的Ste13位点Glu-Ala-Glu-Ala,构建成的皮蝇胶原酶基因分泌型表达载体Hl Collagenase-m/ pGAPZα-A。
5.一种转化子，其特征在于宿主菌被权利要求2或3所述的皮蝇胶原酶突变体表达载体所转化。
6.根据权利要求5所述的转化子，其特征在于所述的宿主菌为毕赤酵母（Pichia pastoris）菌株GS115。
7.一种生产皮蝇胶原酶突变体的方法，其特征在于培养权利要求5或6所述的转化子，收集其分泌产物。
8.一种皮蝇胶原酶突变体毕赤酵母表达产物的纯化方法，其特征在于直接从含权利要求4所述的分泌型表达载体Hl Collagenase-m/ pGAPZα-A的毕赤酵母（Pichia pastoris）菌株GS115的发酵液中分离纯化皮蝇胶原酶突变体：离心收集上清液，以氢氧化钠调节pH值，过阴离子交换层析柱，收集目标洗脱峰，得到纯度约70%的皮蝇胶原酶突变体粗品，透析除盐，超滤浓缩，过分子筛层析柱，收集目标蛋白，得到纯度大于95%的皮蝇胶原酶突变体蛋白。
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