CN117126754A - 重组i型胶原蛋白毕赤酵母工程菌及其构建方法与应用 - Google Patents
重组i型胶原蛋白毕赤酵母工程菌及其构建方法与应用 Download PDFInfo
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Abstract
本发明公开了一种重组I型胶原蛋白毕赤酵母工程菌及其构建方法与应用。合成I型人源化胶原蛋白α1链成熟肽的基因序列，插入载体的α‑factor分泌信号肽Kex2蛋白酶识别位点下游，构建重组质粒pPICZalphaA‑col(I)α1，导入毕赤酵母X‑33，筛选得到重组菌株。本发明还提供一种应用该重组菌株制备I型胶原蛋白的方法，其中提供了一种改良型发酵培养基和高密度发酵方法，表达水平达到6g/L以上，纯化制备的重组I型胶原蛋白纯度达到98%以上，与人I型胶原蛋白氨基酸序列相应部分100％相同。生产过程绿色环保，产品具有更高的生物相容性和生物安全性，产品可以应用于医美、医疗器械及医用生物材料等广泛领域。
Description
技术领域
本发明涉及基因工程技术领域，更具体地，本发明涉及一种重组I型胶原蛋白毕赤酵母工程菌及其构建方法与应用。
背景技术
胶原蛋白是哺乳动物体内含量最多、分布最广的功能性蛋白，约占动物机体蛋白质总量的25％～30％。其中I型胶原蛋白在目前已发现的28中胶原蛋白中含量最高，占全部胶原蛋白含量的80％～90％左右，主要分布于皮肤、角膜、肌腱等部位，对维护细胞、组织等的正常生理功能和损伤修复起着重要作用。在胶原蛋白家族的众多成员中，I型胶原蛋白是组织分布最为广泛和研究最为透彻的胶原蛋白，目前已应用于美容化妆、保健、医疗器械和生物医学材料等领域。
传统生产胶原蛋白的方法是利用酸、碱、酶解法处理猪、牛、鱼等动物来源组织，提取长度不等的混合胶原肽段，生物活性差，并且有病毒感染和免疫排斥等潜在风险。而利用基因工程技术在宿主细胞表达的重组胶原蛋白具有组分一致、质量稳定、安全性好的优点，尤其适合在医疗器械领域等高端领域的应用。
国内外科研人员利用毕赤酵母系统分泌表达重组I型胶原蛋白进行了大量研究，Myllyharju等(2000年)开展了甲基酵母(Pichia pastoris)胞内表达人I、Ⅱ、Ⅲ型胶原蛋白的研究。美国Fibrogen公司与日本国家传染病研究中心(2005年)合作，利用毕赤酵母X-33表达长度101aa的重组I型人源化胶原蛋白。Takahiro等(2020年)根据人类I型胶原蛋白α1链716到779的蛋白质序列，融合4个重叠胶原片段构建胶原蛋白多肽(RCPhC1)。Pia等(2019年)在Ⅰ型人类胶原蛋白α链的541至940aa两侧分别连接(Pro-Gly-Pro)9序列构建GelMP，其与动物明胶有相似的生物相容性和细胞粘附性。Tsuneyuki等(2019年)在毕赤酵母表达人类I型胶原α1链的(RGD)12基序分子。
国内胶原蛋白厂商大多采用大肠杆菌发酵表达胶原蛋白，山西锦波生物医药股份有限公司在专利CN109293783B公开利用大肠杆菌表达系统表达I型人源化胶原蛋白片段，国内专利CN106046151A公开一种大肠杆菌表达人源化可溶高活性I型胶原蛋白片段及其制备方法。大肠杆菌表达系统普遍存在以下缺点：目标蛋白一般以包涵体形式表达增加纯化及除去杂质的难度；产品中残留的内毒素会产生致热源容易引发过敏反应、翻译后加工修饰体系不完善使得表达产物生物活性较低等。
国内利用毕赤酵母系统重组表达I型人源化胶原蛋白片段的研究逐年增多，陕西慧康生物科技有限责任公司在专利CN106554410A公开利用毕赤酵母重组表达I型人源化胶原蛋白片段；西安华澳丽康生物工程有限公司在专利CN102020716B公开在毕赤酵母SMD1168表达全长839个氨基酸的胶原蛋白片段组合，其N端为人Ⅲ型胶原蛋白肽段，C端为人I型胶原蛋白肽段，并用谷氨酸和苯丙氨酸连接起来。西安德诺海思医疗科技有限公司在专利CN113667709A公开了一种在毕赤酵母表达I型胶原蛋白66.2KDa片段的发酵方法，可以减少重组人源化化胶原蛋白在发酵过程发生降解，进行稳定的工业化生产。
迄今，有2家国内企业报道重组表达I型人源化胶原蛋白α1链成熟肽全长序列。其中西安巨子生物基因技术股份有限公司在专利CN103725622A公开毕赤酵母表达改造的I型胶原蛋白α1链成熟肽。江苏悦智生物医药有限公司在专利CN109988234A公开了一种酵母重组I型人源化胶原α1链蛋白、合成方法及其应用，从氨基端依次包含：氨基端亲和纯化标签、I型人源化胶原蛋白α1链成熟肽链以及羧基端亲和纯化标签。两端设计的双特异性亲和纯化标记虽然有利于该重组蛋白的纯化、检测及全长鉴定。如果产品不去除纯化标记，在医学领域应用中存在引发过敏反应的潜在风险；而增加酶切工序除去纯化标记则会使生产工艺复杂化，造成生产成本的提高。
I型人源化胶原蛋白α1链成熟肽含有1057个氨基酸，具有更高的分子量，拥有独特的生理生化特性，是一种前景广阔的医学再生材料。利用毕赤酵母分泌表达与人I型胶原蛋白氨基酸序列相应部分100％相同的重组I型人源化胶原蛋白成熟肽，可以填补目前的国内技术空白。
发明内容
本发明旨在提供一种重组I型胶原蛋白成熟肽的毕赤酵母毕赤酵母工程菌及其构建方法与应用，实现I型胶原蛋白成熟肽在毕赤酵母的高效生产，并获得纯化产品。
为解决上述技术问题，本发明提供如下技术方案：
一种重组I型胶原蛋白毕赤酵母菌株：将I型胶原蛋白基因串联至pPICZalphaA的α-factor信号肽Kex2蛋白酶识别位点下游，目标蛋白分泌至细胞外。
特别的，所述Ⅰ型胶原蛋白为Ⅰ型人源化胶原蛋白α1链成熟肽。
特别的，所述Ⅰ型人源化胶原蛋白α1链成熟肽的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示。利用毕赤酵母分泌表达的目标蛋白与人I型胶原蛋白氨基酸序列相应部分100％相同，不含纯化标签蛋白和其它多余的氨基酸。
特别的，本发明提供了一种重组I型人源化胶原蛋白α1链成熟肽的毕赤酵母工程菌株，所述菌株保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号是CGMCCNo.24534。
本发明还提供一种分泌表达重组I型人源化胶原蛋白α1链成熟肽毕赤酵母工程菌的构建方法：合成的I型人源化胶原蛋白基因串联至pPICZalphaA的α-factor信号肽Kex2蛋白酶识别位点下游。
特别地，上述构建方法由以下步骤实现：
(1)重组I型人源化胶原蛋白α1链成熟肽的氨基酸序列为SEQ ID NO.1。
(2)重组I型人源化胶原蛋白α1链成熟肽的核苷酸序列根据毕赤酵母偏好性密码子进行优化，通过全基因合成I型人源化胶原蛋白α1链成熟肽的基因序列，核苷酸序列如SEQ ID NO.2所示。
(3)双酶切改造载体pPICZalphaA，将目的基因串联至载体α-factor signal的Kex2蛋白酶识别位点下游，构建重组质粒pPICZalphaA-col(Ⅰ)α1。
(4)重组质粒pPICZalphaA-col(Ⅰ)α1用限制性内切酶PmeI线性化处理，将其转化入毕赤酵母X-33感受态细胞中，Zeocin抗性筛选阳性重组子，得到重组I型人源化胶原蛋白α1链成熟肽毕赤酵母工程菌株。所述转化可以选电击转化。
上述构建步骤获得的重组I型人源化胶原蛋白α1链成熟肽毕赤酵母工程菌株，能利用毕赤酵母自身的Kex2蛋白酶切除重组目标蛋白N-端信号肽，分泌表达完整的重组I型人源化胶原蛋白成熟肽。
本发明还提供一种应用上述重组I型人源化胶原蛋白α1链成熟肽毕赤酵母工程菌株制备I型胶原蛋白的方法，包括发酵培养方法和蛋白纯化方法：
1.发酵培养方法：
本发明提供了一种改良型发酵培养基，包含以下浓度的组分：85％磷酸24～28ml/L，二水合硫酸钙0.65～1.15g/L，硫酸钾16.2～20.2g/L，二水合硫酸镁12.9～16.9g/L，氢氧化钾3.53～4.95g/L，甘油30～50g/L，PTM13.5～5.5ml/L，抗坏血酸50～200mg/L，赖氨酸50～300mg/L，山梨醇20～100g/L，肌醇5～20mg/L，叶酸5～30mg/L，泛酸钙2～20mg/L，PH5.0～7.0。
优选的，一种改良型发酵培养基包含以下浓度的组分：85％磷酸26.7ml/L，二水合硫酸钙0.93g/L，硫酸钾18.2g/L，二水合硫酸镁14.9g/L，氢氧化钾4.13g/L，甘油40g/L，PMT14.0ml/L，抗坏血酸20mg/L，赖氨酸150mg/L，山梨醇36.4g/L，肌醇10mg/L，叶酸20mg/L，泛酸钙10mg/L，PH 6.0。
本发明还提供了一种高密度发酵方法：菌体湿重达到200-300g/L时，进入甲醇诱导阶段，开始补加甲醇(100ml甲醇加1.2ml PMT1)，调节PH为5.5±0.3，温度为27±2℃，前12小时控制甲醇补加速度2～4ml/h/L，然后调整甲醇补加速度流速至5～7ml/h/L保持10-14小时，最后甲醇补加速度保持8～12ml/h/L继续诱导70-74小时。
优选的，菌体湿重达到200-300g/L时，开始补加甲醇(100ml甲醇加1.2ml PMT1)，前12小时控制补加速度3ml/h/L，然后调整流速至6ml/h/L保持12小时，最后10ml/h/L继续诱导72小时。
优选的，高密度发酵包括以下步骤：
(1)将存的甘油菌液接种于1500ml YPD培养基，30℃，200rpm培养至OD＝4.0～6.0制备一级种子。
(2)甘油培养阶段：将种子液转接至装有20L改良培养基的发酵罐，设定发酵初始温度30℃，PH5.0，转速500rpm，通气20L/min，培养20h左右。
(3)甘油流加阶段：培养20h左右开始甘油流加阶段，以18ml/h/L的速率补加50％(w/v)甘油，甘油中添加微量元素PTM1(浓度为12ml/L)，直到菌体湿重达到220-260g/L。
(4)甲醇诱导阶段：甘油流加结束30min开始补加甲醇(100ml甲醇加1.2ml PMT1)，甲醇诱导阶段的前12h控制补加速度3ml/h/L，然后调整流速至6ml/h/L保持12h，最后调整至8～9ml/h/L诱导48h结束发酵。
进一步，本发明利用上述改良无机培养基进行重组工程菌株的高密度发酵，能显著提高目标蛋白表达水平，能够实现I型人源化胶原蛋白规模化生产。
2.蛋白纯化方法：
重组I型胶原蛋白α1链成熟肽的蛋白纯化方法包括以下步骤：离心收集发酵上清液，加入饱和硫酸铵溶液获得沉淀，柠檬酸缓冲液溶解沉淀，进行阳离子交换层析，得到I型胶原蛋白。
优选的，蛋白纯化方法包括以下步骤：
(1)离心收集发酵上清液，加入饱和硫酸铵溶液至饱和度为30％，室温静置30min，5000rpm，离心10min，收集沉淀。
(2)25mM柠檬酸盐、50mM氯化钠、pH6.2的缓冲液溶解沉淀，利用Capto SP ImpRes(Cytiva公司产品)进行阳离子交换层析，收集的洗脱组分经30KD超滤膜包置换到20mM PBS(PH7.0)缓冲液，得到所述重组I型胶原蛋白α1链成熟肽。
本发明制备的I型人源化胶原蛋白为完整的重组胶原蛋白，与I型人胶原蛋白氨基酸序列相应部分100％相同，没有任何冗余氨基酸，与明胶和常规的重组胶原片段相比，拥有更完整的结构和生物学性能，可以广泛应用于化妆品、医疗器械、医学材料、组织工程、营养保健和细胞培养等领域。
与现有技术相比，本发明的有益效果是：
(1)本发明根据毕赤酵母偏好性密码子进行优化，消除了不常用密码子及发夹结构，通过全基因合成I型人源化胶原蛋白α1链成熟肽的基因序列，更适于在毕赤酵母中稳定表达。
(2)本发明独创性设计构建重组质粒，目的基因插入α-factor分泌信号肽Kex2蛋白酶识别位点下游，利用毕赤酵母自身的Kex2蛋白酶切除重组目标蛋白N-端信号肽，得到完整的重组胶原蛋白。所得重组胶原蛋白与I型人胶原蛋白氨基酸序列相应部分100％相同，没有任何冗余氨基酸，与明胶和常规的重组胶原片段相比，拥有更完整的结构和生物学性能，是有广阔应用前景的生物医用材料。
(3)本发明利用真核表达系统毕赤酵母分泌表达重组I型人源化胶原蛋白，无病毒隐患，具有更高的生物相容性和生物安全性。克服了传统动物源性胶原蛋白的病毒感染、免疫排斥等潜在风险，还可以避免大肠杆菌表达系统的纯化困难、易产生致热源等缺点。
(4)本发明利用改良培养基进行高密度发酵，克服标准无机培养基的不足，发酵周期短，操作简单方便，能够显著提高重组I型胶原蛋白α1链成熟肽的表达水平到6g/L以上。
(5)本发明制备重组I型人源化胶原蛋白成熟肽的纯化方法步骤简单，产品纯度达98％以上，可以安全应用于生物医学材料、医疗器械等领域。
附图说明
图1为重组质粒pPICZalphaA-col(Ⅰ)α1图谱。
图2是PCR鉴定重组质粒pPICZalphaA-col(Ⅰ)α1的琼脂糖凝胶电泳图谱，其中泳道M为DL5000 DNA Marker，泳道1、2为不同重组质粒的PCR扩增产物。
图3是PCR验证重组子pPICZalphaA-col(Ⅰ)α1/X-33的琼脂糖凝胶电泳图谱。其中泳道M为DL5000 DNA Marker，泳道1、2为不同重组子的PCR扩增产物。
图4是摇瓶筛选重组菌株的SDS-PAGE电泳图谱，其中泳道M为PageRulerTMPrestained Protein Ladder，泳道1～3为不同的重组菌落。
图5是不同培养基摇瓶发酵样品对比的SDS-PAGE图谱，其中泳道M为PageRulerTMPrestainedProtein Ladder，泳道1～3为改良培养基发酵液样品，泳道4～6为标准无机盐培养基发酵液样品。
图6是发酵液纯化样品的SDS-PAGE电泳检测图谱，其中泳道M为PageRulerTMPrestained Protein Ladder，泳道1为纯化样品。
具体实施方式
下面结合具体实施方式解释本发明，参考附图描述的实施例是示例性的，不能理解为对本发明的限制。
以下实施例中的实验方法，如无特别说明，均为常规方法。其中，本发明所选用的毕赤酵母(Pichiapastoris)X-33菌株、表达载体pPICZalphaA均购自北京金赛狮生物制药技术开发有限责任公司。本发明应用的培养基配方如下：
LLB培养基(1L)：酵母提取物5g，蛋白胨10g，氯化钠10g(固体培养基含2％琼脂粉)。
YPD培养基(1L)：酵母提取物10g，蛋白胨20g，葡萄糖20g。
YPDS培养基(1L)：酵母提取物10g，蛋白胨20g，葡萄糖20g，1M山梨醇(固体培养基含2％琼脂粉)。
BSM无机培养基配方(1L)：85％磷酸26.7ml，二水合硫酸钙0.93g，硫酸钾18.2g，七水合硫酸镁14.9g，氢氧化钾4.13g，甘油40g，PMT14.35 ml。
PMT1培养基配方(1L)：五水合硫酸铜6.0g，碘化钾0.088g，一水合硫酸锰3.0g，二水合钼酸钠0.2g，硼酸0.02g，六水合氯化钴0.5g，氯化锌20.0g，七水合硫酸亚铁65.0，生物素0.2g，浓硫酸5.0ml。
在本发明中，提供了一种重组I型人源化胶原蛋白α1链成熟肽的毕赤酵母工程菌株(Pichiapastoris)，所述菌株保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号是CGMCC No.24534，保藏日期：2022年3月15日，地址朝阳区北辰西路1号院3号中国科学院微生物研究所，分类命名：巴斯德毕赤酵母(Pichiapastoris)。
以下为毕赤酵母基因工程菌构建、发酵及纯化制备重组I型胶原蛋白的具体过程。
1、基因合成及重组质粒的构建
(1)人工合成I型人源化胶原蛋白α1链成熟肽的基因序列：在不改变I型人源化胶原蛋白α1链成熟肽(NCBI Reference Sequence:NP_000079.2)的氨基酸序列(SEQ ID NO.1所示)情况下，根据毕赤酵母偏好性密码子，权衡GC含量、密码子使用频率和RNA酶的剪接位点、RNA稳定反式作用元件等因素，并剔除了BglⅡ、BamHI、Not I、PmeI、Sac I、Stu I等限制性内切酶位点，由南京金斯瑞生物科技有限公司全合成I型人源化胶原蛋白α1链成熟肽的基因序列col(Ⅰ)α1，核苷酸序列如SEQ ID NO.2所示。
(2)扩增目的基因：以合成基因col(Ⅰ)α1为模板，利用引物P1、P2进行PCR扩增，其中P1的基因序列如SEQ ID NO.3所示，P2的基因序列如SEQ ID NO.4，胶回收目的基因片段。
(3)构建重组质粒：分别用XhoI和XbaI双酶切PCR扩增产物col(Ⅰ)α1和载体质粒pPICZalphaA，经T4 DNA连接酶于16℃连接4h，连接产物热击转化至感受态细胞DH10b，涂布于Zeocin抗性的低盐LLB平板。挑取单菌落提取质粒进行PCR鉴定，如图2所示，扩增条带与I型胶原蛋白α1链成熟肽基因(3196bp)大小相符，得到阳性重组质粒pPICZalphaA-col(Ⅰ)α1。
2、制备毕赤酵母X-33感受态细胞
(1)挑取酵母X-33单菌落，接种至含有5mL的YPD液体培养基试管，30℃、200rpm振荡培养过夜。
(2)取500μL的过夜培养物接种至含有50mL新鲜YPD液体培养基的三角瓶中，30℃、200rpm振荡培养过夜。
(3)将培养物于4℃，1500×g离心5min，用50mL预冷的无菌双蒸水重悬菌体后离心。
(4)用25mL预冷的无菌双蒸水重悬菌体后离心。
(5)用20mL预冷的1M山梨醇溶液重悬菌体后离心。
(6)用0.5mL预冷的1M山梨醇溶液重悬菌体即为制备的感受态细胞。
3、线性化重组质粒及电转化毕赤酵母X-33
(1)试剂盒提取重组质粒pPICZalphaA-col(Ⅰ)α1，限制性内切酶PmeI进行线性化处理。
(2)取1-10μg线性化质粒加入80μl毕赤酵母X-33感受态细胞中，混合均匀转至2mm电转化杯中，冰浴5min。
(3)电转仪预热后设置参数(1.5KV，200Ω，25μF)进行电转。
(4)电转结束立即往转化杯中加入1ml预冷的1M山梨醇溶液混匀，30℃静置1h。
(5)取200μl菌液涂布于Zeocin抗性(100μg/ml)的YPDS平板，30℃培养2-5天，直到有单菌落出现。
4、重组子的PCR验证
挑取YPDS平板生长出的单菌落提取基因组DNA，利用引物5′AOX1和3′AOX1进行PCR验证。PCR反应体系为50μl，反应组分包括：10×Taqbuffer 5μl，dNTPs 4μl，上下游引物各1μl，模板2μl，Taq聚合酶1μl，灭菌双蒸水36μl。PCR扩增条件为：95℃预变性5min；95℃变性30s，56℃退火30s，72℃延伸3min，30个循环；72℃延伸10min。琼脂糖凝胶电泳结果如图3所示，其中包含目的基因的条带约是3800bp，宿主菌AOX1基因条带长度是2200bp左右。
5、基因工程菌株的诱导表达
(1)挑取阳性重组子单菌落于50mLYPD培养基中，30℃、200rpm振摇过夜培养进行活化。
(2)1500g离心5min收集菌体，重悬菌体于50mLBMGY培养基中，使起始浓度OD600＝1.0左右，30℃、200rpm下培养18～20h。
(3)调节温度为28℃，每隔24小时向培养基中添加甲醇至终浓度为1％进行诱导表达，甲醇诱导96h。离心收集发酵上清液进行SDS-PAGE电泳检测，图4结果显示3个重组菌落都有与预期分子量130KDa相符的表达条带，但是表达量有所不同。经筛选获得高表达量的重组菌株。
6、不同发酵培养基摇瓶对比实验
(1)保存的甘油菌液接种于50mLYPD培养基中，30℃、200rpm振摇过夜培养进行活化。
(2)1500g离心5min收集菌体，分别重悬菌体于一种改良型发酵培养基和标准无机盐培养基(BSM无机培养基)中，PH 5.0，使起始浓度OD600＝1.0左右，各做3个重复实验。30℃、200rpm下培养18～20h。
(3)调节温度为28℃，每隔24小时向培养基中添加甲醇(100ml甲醇加1.2ml PMT1)至终浓度为1％进行诱导表达，甲醇诱导96h。上述改良型发酵培养基包含以下浓度的组分：85％磷酸26.7ml/L，二水合硫酸钙0.93g/L，硫酸钾18.2g/L，二水合硫酸镁14.9g/L，氢氧化钾4.13g/L，甘油40g/L，微量元素PMT14.0ml/L，抗坏血酸100mg/L，赖氨酸150mg/L，山梨醇36.4g/L，肌醇10mg/L，叶酸20mg/L，泛酸钙10mg/L，PH 6.0。
离心收集发酵上清液进行SDS-PAGE电泳检测，图5结果显示改良型发酵培养基的目标蛋白表达量明显提高。采用灰度分析法对SDS-PAGE结果图谱进行分析，3个改良型发酵培养基摇瓶发酵液的灰度平均值是标准无机盐培养基的3.18倍。
7、重组菌株的高密度发酵培养
(1)将保存的甘油菌液接种于1000ml YPD培养基，30℃，200rpm培养至OD＝2～6制备一级种子。
(2)按5％接种量将一级种子液转接至装有20L培养基的的发酵罐，设定发酵初始温度30℃，PH5.0，转速500rpm，通气20L/min。
(3)培养20h左右开始甘油流加阶段，以18ml/h/L的速率补加50％(w/v)甘油，甘油中添加微量元素PTM1(浓度为12ml/L)，直到菌体湿重达到220～260g/L。
(4)甘油流加结束30分钟开始补加甲醇(100ml甲醇加1.2ml PMT1)，甲醇诱导阶段的前12h控制补加速度3ml/h/L，然后调整流速至6ml/h/L保持12h，最后调整至8～9ml/h/L诱导48h结束发酵，离心收集发酵上清液。利用Bradford方法测定使用改良型培养基的重组蛋白表达水平达到6.25g/L，明显高于标准培养基的发酵水平。
8、重组I型胶原蛋白的纯化
(1)发酵收获液经离心去除菌体得到上清液，加入饱和硫酸铵溶液至饱和度25％，搅拌充分混匀后，静置30min，离心收集沉淀。
(2)用超纯水将沉淀稀释后，用30KD超滤膜包进行换液，置换到25mM柠檬酸盐、50mM氯化钠、pH6.2的柠檬酸缓冲液中，得到层析上样液。
(3)进行Capto SP ImpRes(Cytiva公司)层析：
平衡：平衡液25mM柠檬酸盐缓冲液、50mM氯化钠、pH6.2，平衡4-5柱体积(CV)；
上样：上述超滤置换液，30-40g/Lresin；洗涤：25mM柠檬酸缓冲液盐、50mM氯化钠、pH6.2，2CV；
洗脱：25mM柠檬酸缓冲液盐、250mM氯化钠、pH6.2，洗脱4CV得目标蛋白组分。
(4)收集的洗脱组分经过30KD超滤膜包换液，目标蛋白置换到20mM PBS(PH7.0)缓冲液中，图6的SDS-PAGE电泳检测结果显示得到的重组I型胶原蛋白α1链成熟肽，与预期分子量130KDa大小相符，纯度达到98％以上。并且该纯化组分可以被胶原蛋白水解酶(Sigma，C5138-25MG)专一性水解。
本发明的内容不限于实施例所列举，本领域普通技术人员通过阅读本发明说明书而对本发明技术方案采取的任何等效的变换，均为本发明的权利要求所涵盖。
序列表
<110> 华北制药集团新药研究开发有限责任公司
<120> 重组I型胶原蛋白毕赤酵母工程菌及其构建方法与应用
<130> 2021-11-05
<160> 4
<170> SIPOSequenceListing 1.0
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ccaggtccta tgggtccatc tggtcctaga ggtttgccag gtccacctgg tgctccaggt 120
cctcaaggtt tccaaggtcc acctggtgaa ccaggagagc ctggtgcttc tggtccaatg 180
ggtcctagag gtccacctgg tccacctggt aaaaacggag atgatggtga agctggtaaa 240
ccaggtagac ctggagagag aggtccacct ggacctcaag gtgctagagg tttgcctggt 300
actgctggtt tgcctggtat gaagggtcat agaggttttt ctggtttgga tggtgctaag 360
ggagatgctg gtccagctgg tcctaaaggt gaaccaggtt ctcctggaga gaacggtgct 420
ccaggtcaaa tgggacctag aggtttgcct ggagagagag gtagaccagg tgctccaggt 480
cctgctggtg ctagaggtaa cgatggtgct actggtgctg ctggtccacc tggaccaact 540
ggtcctgctg gtccacctgg tttcccaggt gctgttggtg ctaaaggtga agctggtcca 600
caaggtccta gaggttctga aggtccacaa ggtgttagag gtgagccagg tccacctggt 660
ccagctggtg ctgctggtcc tgctggtaac ccaggtgctg atggtcaacc aggtgctaag 720
ggtgctaatg gtgctcctgg tattgctggt gctccaggtt ttcctggtgc tagaggtcca 780
tctggtcctc aaggtccagg tggtccacct ggacctaagg gtaattctgg agagcctggt 840
gctccaggtt ctaagggaga tactggtgct aaaggtgaac caggtcctgt tggtgttcaa 900
ggtccacctg gacctgctgg tgaagagggt aaaagaggtg ctagaggtga accaggtcct 960
actggtttgc ctggtccacc tggagaaaga ggtggtcctg gttccagagg tttcccaggt 1020
gctgatggtg ttgctggtcc taagggtcca gctggtgaaa gaggttctcc aggtcctgct 1080
ggtcctaaag gttctccagg tgaagctggt agacctggag aggctggttt gccaggtgct 1140
aagggtttga ctggttctcc aggttctcca ggtcctgatg gtaaaaccgg tccacctgga 1200
ccagctggtc aagatggtag acctggtcca cctggtccac ctggtgctag aggtcaagct 1260
ggtgttatgg gttttccagg tcctaagggt gctgctggtg aacctggtaa agctggagag 1320
agaggtgttc caggtccacc tggagccgtt ggtcctgctg gtaaagatgg tgaagctggt 1380
gctcaaggtc cacctggtcc tgctggacct gctggtgaaa gaggtgagca aggtcctgct 1440
ggttctccag gtttccaagg tttgccagga ccagctggtc cacctggaga ggctggtaaa 1500
cctggagagc aaggtgttcc aggagatttg ggtgctccag gtccttctgg tgctagaggt 1560
gaaagaggtt ttccaggaga gagaggtgtt cagggtccac ctggaccagc cggtccaaga 1620
ggtgctaacg gtgctccagg aaatgacgga gccaaaggag atgctggtgc tcctggtgct 1680
ccaggttctc aaggtgctcc tggtttgcag ggtatgccag gagagagagg tgctgctggt 1740
ttgccaggtc ctaaaggaga tagaggagat gctggtccaa agggtgctga tggttctcct 1800
ggtaaagatg gtgttagagg tttgactggt ccaatcggtc cacctggtcc agcaggtgct 1860
ccaggagata agggagagtc tggtccttcc ggaccagctg gtcctactgg tgctagaggt 1920
gctccaggag atagaggtga acctggtcca cctggacctg ccggtttcgc tggtccacct 1980
ggtgctgatg gtcaacctgg tgctaagggt gaaccaggag atgctggtgc taaaggagat 2040
gccggtccac ctggacctgc cgggccagcc ggtccacctg gtccaattgg taacgttggt 2100
gctcctggtg ctaaaggtgc tagaggttct gccggtccac ctggagctac tggttttcca 2160
ggtgctgctg gtagagtcgg tccacctggt ccttctggta acgccggtcc acctggtcca 2220
cctgggccag ctggtaaaga aggtggtaaa ggtcctagag gtgagactgg tcctgctggt 2280
agaccaggtg aggtcggtcc acctggtcca cctggaccgg ctggagagaa gggttctcct 2340
ggtgctgatg gtcctgctgg tgctccaggt actccaggtc ctcaaggtat tgctggtcaa 2400
agaggtgttg ttggtttgcc aggtcaaaga ggtgaaagag gtttccctgg tttgccaggt 2460
ccttctggag agcctggtaa acaaggtcca tctggtgctt ctggtgaaag aggtccacct 2520
ggacctatgg gtccacctgg tttggctggt ccacctggtg aatctggtag agagggtgct 2580
cctggtgctg agggttctcc aggtagagat ggttctcctg gtgctaaggg agatagaggt 2640
gaaactggtc cagcaggtcc acctggtgct ccaggtgctc ctggtgctcc agggcctgtt 2700
ggtccagctg gtaaatccgg agatagaggt gagaccggac cagctggtcc tgctggtcca 2760
gttggtcctg ttggtgctag aggtccagct ggtcctcaag gtccaagagg agataagggt 2820
gaaactggag agcaaggaga tagaggtatt aaaggacacc gtggtttttc tggtttgcag 2880
ggtccacctg gtccacctgg ttctcctggt gagcagggac cttctggtgc ttctggtcct 2940
gctggtccaa gaggtccacc tggttctgcc ggtgctccag gtaaagatgg tttgaacggt 3000
ttgccaggtc ctatcggtcc acctggacca agaggtagaa ctggagatgc tggtcctgtg 3060
ggtccacctg gtccacctgg tccacctggt ccacctggtc caccttctgc tggttttgat 3120
ttctctttct tgccacaacc acctcaagaa aaagctcacg atggtggtag atactataga 3180
gcttaatcta ga 3192
<210> 3
<211> 36
<212> DNA
<213> 人工合成(Synthetic Sequence)
<400> 3
ctctcgagaa aagacaattg tcttacggtt atgatg 36
<210> 4
<211> 25
<212> DNA
<213> 人工合成(Synthetic Sequence)
<400> 4
gttctagatt aagctctata gtatc 25
Claims (10)
1.一种重组I型胶原蛋白毕赤酵母工程菌，其特征在于：将I型胶原蛋白基因串联至pPICZalphaA的α-factor信号肽Kex2蛋白酶识别位点下游，目标蛋白分泌至细胞外。
2.根据权利要求1所述的重组I型胶原蛋白毕赤酵母工程菌，其特征在于：所述Ⅰ型胶原蛋白为Ⅰ型人源化胶原蛋白α1链成熟肽。
3.根据权利要求2所述的重组I型胶原蛋白毕赤酵母工程菌，其特征在于：所述Ⅰ型人源化胶原蛋白α1链成熟肽的氨基酸序列如SEQ ID NO.1所示，核苷酸序列如SEQ ID NO.2所示。
4.根据权利要求1-3任一所述重组I型胶原蛋白毕赤酵母工程菌，其特征在于：所述菌株保藏在中国微生物菌种保藏管理委员会普通微生物中心，保藏编号是 CGMCC No.24534。
5.一种权利要求1所述重组I型胶原蛋白毕赤酵母工程菌的构建方法，其特征在于：合成的I型人源化胶原蛋白基因串联至pPICZalphaA的α-factor信号肽Kex2蛋白酶识别位点下游。
6.一种应用权利要求1所述工程菌制备I型胶原蛋白的方法，包括发酵培养和蛋白纯化，其特征在于，发酵培养基包含以下浓度的组分：85% 磷酸24～28ml/L，二水合硫酸钙0.65～1.15g/L，硫酸钾16.2～20.2g/L，二水合硫酸镁12.9～16.9g/L，氢氧化钾 3.53～4.95g/L，甘油30～50g/L，PTM1 3.5～5.5ml/L，抗坏血酸50～200mg/L，赖氨酸50～300mg/L，山梨醇 20～100g /L，肌醇5～20mg/L，叶酸5～30mg/L，泛酸钙2～20mg/L，PH 5.0～7.0。
7.根据权利要求6所述的方法，其特征在于，发酵培养基包含以下浓度的组分：85% 磷酸26.7ml/L，二水合硫酸钙0.93g/L，硫酸钾18.2g/L，二水合硫酸镁14.9g/L，氢氧化钾4.13g/L，甘油40g/L，PMT1 4.0ml/L，抗坏血酸100mg/L，赖氨酸150mg/L，山梨醇36.4g /L，肌醇10mg/L，叶酸20mg/L，泛酸钙10mg/L，PH 6.0。
8.根据权利要求6所述的方法，其特征在于，所述发酵培养包括以下步骤：菌体湿重达到220～260 g/L时开始补加甲醇，100ml甲醇加1.2ml PMT1，调节PH为5.5±0.3，温度为27±2℃，前12小时甲醇补加速度为2～4 ml/h/L，然后为5～7 ml/h/L保持10-14小时，最后为8～12ml/h/L保持70-74小时。
9.根据权利要求8所述的方法，其特征在于，所述发酵培养包括以下步骤：菌体湿重达到220～260 g/L时开始补加甲醇，100ml甲醇加1.2ml PMT1，前12小时甲醇补加速度3 ml/h/L，然后为6 ml/h/L保持12小时，最后为10ml/h/L保持72小时。
10.根据权利要求6～9任一所述的方法，其特征在于，所述蛋白纯化包括以下步骤：离心收集发酵上清液,加入饱和硫酸铵溶液获得沉淀，柠檬酸缓冲液溶解沉淀，进行阳离子交换层析，得到I型胶原蛋白。
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