CN116334015A - 一种用于重组胶原修饰的脯氨酸羟化酶筛选方法及应用 - Google Patents
一种用于重组胶原修饰的脯氨酸羟化酶筛选方法及应用 Download PDFInfo
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Abstract
本发明涉及一种用于重组胶原修饰的脯氨酸羟化酶筛选方法及应用,将连接有脯氨酸羟化酶的重组质粒转入宿主菌中,在LB培养基中直接添加脯氨酸和辅助因子共培养发酵,加入IPTG诱导表达,用氯胺T方法检测酶活,高通量筛选出具有酶活的脯氨酸羟化酶;再将连接有步骤S1中筛选出的脯氨酸羟化酶与重组胶原的重组质粒转入宿主菌中,对菌株进行诱导表达,通过Western blot对重组胶原蛋白的羟化修饰作用进行检测,所提供的方法能够快速筛选对于重组胶原蛋白具有修饰作用的脯氨酸羟化酶,并体现其修饰能力。
Description
技术领域
本发明属于生物技术领域,具体涉及一种用于重组胶原修饰的脯氨酸羟化酶筛选方法及应用。
背景技术
反式-4-L-羟脯氨酸(反式-4-Hyp)是合成碳青霉烯类抗生素、血管紧张素转换酶抑制剂和解痉剂等药物的重要手性构件。目前反式-4-L-羟脯氨酸来源于胶原蛋白的酸解,存在产率低,环境污染严重等缺点。脯氨酸羟化酶(P4H)是将游离L-脯氨酸羟基化为反式-4-L-羟脯氨酸的关键酶。近年来,在重组菌当中表达反式-4-Hyp因其产率高,环境影响小等优点而受到了越来越多的关注。稳定生产重组P4H不仅对研究酶的结构和功能具有重要意义,而且对于定向进化改进酶活性机制的研究也具有重要意义。
反式-4-L-羟脯氨酸是胶原蛋白中特有的亚氨基酸,只能由脯氨酸羟化酶将脯氨酸羟化之后形成反式-4-L-羟脯氨酸。因此反式-4-L-羟脯氨酸是直接测定生物样品中胶原含量的重要标记物,测定反式-4-L-羟脯氨酸含量也成为了检测脯氨酸羟化酶活性的重要指标。利用高效液相色谱(HPLC)对酸解后蛋白样品进行检测是最常见的方法之一,但是HPLC耗时并且昂贵,对设备要求高。例如:专利CN106834244A和CN109715817A:利用HPLC检测筛选多种脯氨酸羟化酶测定酶活时选用的突变体。该方法成本较高,耗时较长。
利用紫外分光光度法代替HPLC能更加快速便捷的测定反式-4-L-羟脯氨酸含量,其原理是氯胺T将反式-4-L-羟脯氨酸氧化为吡咯环后,加入高氯酸与过量的氯胺T反应,再与对二氨基苯甲醛反应产生红色化合物,最后检测吸光值。近年来测定重组菌中反式-4-L-羟脯氨酸含量常用方法为测定全细胞酶活,该方法需要诱导菌株,收集菌体,再利用酶反应缓冲溶液进行反应,最后利用紫外分光光度法对上清进行比色检测。该方法较为繁琐,在筛选多种脯氨酸羟化酶时,耗时长,实验费用偏高。而针对于胶原蛋白修饰的羟化酶筛选鲜有报道,CN111334512A中提及脯氨酸羟化酶与赖氨酸羟化酶与胶原蛋白共表达,并且利用ELISA检测试剂盒检测了羟脯氨酸含量与羟赖氨酸含量,该方法周期长,操作繁琐,同时未提及酶活性的检测。
发明内容
本发明的目的在于克服现有技术中存在的缺陷,提供一种简便高效的用于重组胶原修饰的脯氨酸羟化酶筛选方法,并同时提供其应用。
为解决上述问题,本发明所采取的技术方案是:
本发明一方面提供了一种用于重组胶原修饰的脯氨酸羟化酶筛选方法,其包括如下步骤:
其包括如下步骤:
S1:将连接有脯氨酸羟化酶的重组质粒转入宿主菌中,在LB培养基中直接添加脯氨酸和辅助因子共培养发酵,加入IPTG诱导表达,用氯胺T方法检测酶活,高通量筛选出具有酶活的脯氨酸羟化酶;
S2:将连接有步骤S1中筛选出的脯氨酸羟化酶与重组胶原的重组质粒转入宿主菌中,对菌株进行诱导表达,通过Western blot对重组胶原蛋白的羟化修饰作用进行检测。
作为本发明的一些优选实施方式,所述辅助因子选自FeSO4、抗坏血酸和α-酮戊二酸。
作为本发明的一些优选实施方式,所述FeSO4浓度为75mg/L、抗坏血酸浓度为10mg/L和α-酮戊二酸浓度为35mg/L。
作为本发明的一些优选实施方式,所述脯氨酸的量为5g/L。
作为本发明的一些优选实施方式,所述S1中的重组质粒为pACYCDuet-P4H,所述S2中的重组质粒为ACYCDuet-P4H-RC。
作为本发明的一些优选实施方式,所述宿主菌为大肠杆菌BL21。
作为本发明的一些优选实施方式,所述S1的操作如下:
培养含有PACYCDuet-P4H的BL21菌液在OD600处吸光值为0.8-1.0时加入0.5mMIPTG,28℃诱导,诱导24h,诱导完成后,离心取350μL上清液,加入150μL氯胺T溶液,避光静置20min,加入150μL显色剂,水浴60℃,20min,最后冷却至室温,取200μL置于酶标板中,在OD560测得吸光值,计算酶活。
作为本发明的一些优选实施方式,所述S2的操作如下:
对pACYCDuet-1-P4H-RC的BL21(DE3)菌株进行诱导表达,24h取2mL菌液,离心收集菌体,加入300μL 1X蛋白loading Buffer重悬菌体,金属浴95℃,5min后进行Western Blot验证。
本发明另一方面提供了所述的筛选方法在筛选用于重组胶原修饰的脯氨酸羟化酶中的应用。
采用上述技术方案所产生的有益效果在于:
本发明所提供的方法,在LB培养基中添加适量的辅助因子(抗坏血酸、FeSO4、α-酮戊二酸),能有效的提高脯氨酸羟化酶的活性,减少检测步骤和误差;同时结合WesternBlot,检测筛选羟化酶对于重组胶原蛋白的羟化修饰能力。所提供的方法能够快速筛选对于重组胶原蛋白具有修饰作用的脯氨酸羟化酶,并体现其修饰能力。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案,下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍。
图1是本发明实施例1不同来源的脯氨酸羟化酶活性检测的结果示意图;
其中,a为诱导4h结果图;b为诱导8h结果图;c为诱导12h结果图;d为诱导16h结果图;e为诱导20h结果图;f为诱导14h结果;A为pACYCDuet-1空载体;B为PACYCDuet-Hy305;C为PACYCDuet-Hy257;D为PACYCDuet-Hy593;E为PACYCDuet-Hy243;
图2是本发明实施例1不同来源的脯氨酸羟化酶对重组胶原蛋白1206片段羟基化修饰Western Blot结果图;
其中,a为1206菌株诱导4h Western Blot图;b为1206菌株诱导24h West ernBlot图;c为pACYCDuet-1-Hy593-1206诱导0-24hWestern Blot图;A为pACY CDuet-1-Hy305-1206;B为pACYCDuet-1-Hy257-1206;C为pACYCDuet-1206;D为pACYCDuet-1-Hy593-1206;E为pACYCDuet-1-Hy243-1206。
图3是本发明实施例2不同来源的脯氨酸羟化酶对重组胶原蛋白1880NNC片段羟基化修饰Western Blot结果图;
其中,a为1880NNC菌株诱导4hWestern Blot图;b为1880NNC菌株诱导24hWesternBlot图;c为pACYCDuet-1-Hy593-1880NNC诱导0-24hWestern Blot图;A为pACYCDuet-1-Hy305-1880NNC;B为pACYCDuet-1-Hy257-1880NNC;C为pACYCDuet-1880NNC;D为pACYCDuet-1-Hy593-1880NNC;E为pACYCDuet-1-Hy243-1880NNC。
图4是实施例3PACYCDuet-1-P4H-1206羟脯氨酸含量检测结果图;
其中,a为Western Blot结果图;b为强度氨酸浓度图;A为1206;B为Hy593-1206;C为Hy257-1206;
图5是pACYCDuet-1-Hy593-1206和pACYCDuet-1-Hy593-1880NNC不同诱导时间羟脯氨酸含量;
其中,a为Hy593-1206不同诱导时间的Western Blot图;b为Hy593-1206不同诱导时间的羟脯氨酸浓度图;c为Hy593-1880NNC不同诱导时间的Western Blot图;
图6是pACYCDuet-1-Hy593-1206羟基化位点检测图;
图7是pACYCDuet-1-Hy593-1880NNC羟基化位点检测图;
图8为Hy593在不同浓度辅助因子诱导下的活性对比图;
图9为三种辅助因子浓度对Hy593活性影响图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚,下面结合具体实施例对发明进行清楚、完整的描述。
本部分所用的实验物品来源如下:
1.1菌株和质粒
本部分实施例所用的宿主菌为大肠杆菌BL21(DE3),表达载体pACYCDuet-1,表达质粒见表1,质粒由河北科技大学细胞研究工程室构建。
表1高通量筛选表达质粒
1.2主要试剂
羟脯氨酸含量检测试剂盒、L-脯氨酸、氯霉素、异丙基硫代半乳糖苷(Isopropylβ-D-Thiogalactoside,IPTG)、BCA蛋白浓度测定试剂盒、甘氨酸、脱脂奶粉、NaCl,购自索莱宝有限公司;胰蛋白胨、酵母提取物,OXOID公司产;SDS-PAGE蛋白上样缓冲液、超敏ECL化学发光试剂盒,购自碧云天有限公司;预染蛋白Marker,Throm公司产;聚偏二氟乙烯膜(polyvinylidene fluoride,PVDF),购自默克公司。
1.3培养基
LB培养基:胰蛋白胨(10g/L);酵母提取物(5g/L);NaCl(10g/L),按照此配比配制液体培养基。
1.4仪器与设备
恒温摇床振荡器,上海一恒科学仪器有限公司产;电泳凝胶成像分体系统,Biored公司产,电泳槽及电泳仪,北京六一生物科技有限公司产,酶标仪,Throm公司产。
实施例1
用于重组胶原修饰的脯氨酸羟化酶的筛选方法,包括如下步骤:
S1:将连接有脯氨酸羟化酶的重组质粒PACYCDuet-Hy305、PACYCDuet-Hy257、PACYCDuet-Hy593和PACYCDuet-Hy243,以及空载体pACYCDuet-1分别转入宿主菌BL21中,分别在添加有L-脯氨酸5g/L、FeSO4 75mg/L、抗坏血酸10mg/L和α-酮戊二酸35mg/L的LB培养基中培养发酵,将含有的BL21菌液在OD600处吸光值为0.8-1.0时加入0.5mM IPTG,28℃诱导,每隔4h取2mL菌液,离心取350μL上清液,加入150μL氯胺T溶液,避光静置20min,加入150μL显色剂,水浴60℃,20min,最后冷却至室温,取200μL置于酶标板中,在OD560测得吸光值,以pACYCDuet-1测量结果作为本底数值,计算脯氨酸羟化酶的酶活,具体见图1,经筛选Hy257酶和Hy593酶具有酶活性。
S2:将连接有脯氨酸羟化酶与重组胶原的重组质粒pACYCDuet-1-Hy305-1206、pACYCDuet-1-Hy257-1206、pACYCDuet-1-Hy593-1206、pACYCDuet-1-Hy243-1206以及空白重组质粒pACYCDuet-1206转入BL21(DE3)中,对菌株进行诱导表达,每隔4h取2mL菌液,离心收集菌体,加入300μL 1X蛋白loading Buffer重悬菌体,金属浴95℃,5min后进行WesternBlot验证,结果见图2,结果表明仅有Hy593酶能够对共表达的重组胶原进行修饰。
考虑到前期酶的表达量会对催化效果有制约因素,因此筛选时间以12-24h为宜。
实施例2
步骤S1同实施例1。
S2:将连接有脯氨酸羟化酶与重组胶原的重组质粒pACYCDuet-1-Hy305-1880NNC、pACYCDuet-1-Hy257-1880NNC、pACYCDuet-1-Hy593-1880NNC、pACYCDuet-1-Hy243-1880NNC以及空白重组质粒pACYCDuet-1880NNC转入BL21(DE3)中,对菌株进行诱导表达,每隔4h取2mL菌液,离心收集菌体,加入300μL 1X蛋白loading Buffer重悬菌体,金属浴95℃,5min后进行Western Blot验证,结果见图3,结果表明仅有Hy593酶能够对共表达的重组胶原进行修饰。
试验例1
为了对于实施例1的筛选方法进行准确性验证,本试验对分别含有pACYCDute-1206、pACYCDute-Hy593-1206、pACYCDute-Hy257-1206的菌株诱导表达,纯化表达的重组蛋白,利用6mol HCl将蛋白浓度调整一致后,烘箱110℃酸解4-6h,将酸解液PH调整至中性,体积调整一致后,取350μL酸解液,加入150μL氯胺T溶液,避光静置20min,加入150μL显色剂,水浴60℃,20min,最后冷却至室温,取200μL置于酶标板中,在OD560测得吸光值,计算羟脯氨酸含量,具体结果见图4。
结果显示,如图4a,只有Hy593可以羟化重组胶原1206。将纯化得到的蛋白酸解后测定羟脯氨酸的含量,如图4b所示,只有与Hy593羟化酶共表达的重组胶原1206中检测出羟脯氨酸只表达1206、与羟化酶Hy257共表达的1206中均未检测出羟脯氨酸。
试验例2
利用1206和1880NNC片段,察Hy593羟化重组胶原的能力,纯化诱导4h、12h、24h三个时间点的重组胶原1206和1880NNC分别进行Western blot,具体见图5,重组胶原1206随着诱导时间的延长,羟化程度逐步增高,与诱导4h相比,诱导12h后条带密度增加,说明羟化的1206量增多,诱导12h后,条带上移幅度加大,说明羟化程度增高,在对于重组1880NNC片段的羟化中,虽然在Hy593羟化酶作用下,蛋白条带均有上移,但是随着诱导时间的增长,蛋白条带位置并未出现明显变化。纯化与Hy593共表达的重组蛋白1880NNC,在诱导4h、12h和24h时蛋白浓度分别约为0.12mg/mL、0.13mg/mL和0.12mg/mL。
利用质谱对1206与1880NNC进行了羟基化位点检测,图6为质谱检测到的羟化的1206羟化位点,可以看到有28个位点被羟基化形成了羟脯氨酸,羟基化位点集中在中部位置。图7为质谱检测到的羟化的1880NNC羟化位点,结果表明,1880NNC序列只有13个位点被羟化成为羟脯氨酸,其羟化位点与1206羟化位点重合,但其羟化程度仅占1206序列羟化程度的46.4%。表明Hy593羟化短片段的能力要高于羟化长片段的能力,这与测定羟脯氨酸含量的结果是一致的。
试验例3:辅助因子的正交试验
为提高高通量筛选体系的筛选稳定性,对FeSO4、抗坏血酸和α-酮戊二酸三种辅助因子浓度进行摸索,筛选最佳添加浓度。选取五个水平,正交因素水平表如表2所示。按照表3浓度,添加到LB-L-脯氨酸培养基,再检测羟脯氨酸酶活性。
表2正交水平因素表(浓度单位mg/L)
表3L25(3 5)正交表(浓度单位mg/L)
正交实验设为25组,每组三个重复,利用96孔深孔板进行正交实验,建立了高通量筛选体系:
1)在深孔板每孔中加入3颗直径为2mm震荡珠。
2)在深孔板每个孔中加入700μL含氯霉素抗性的LB培养基,每个孔中加入50uLHy593菌液,深孔板中含有3孔未诱导菌液与3孔只加IPTG诱导菌液。
3)37℃,300rpm,震荡培养12h。
4)加入终浓度为0.5mM IPTG,将温度调整至28℃,诱导24h。
5)诱导完成后,取100μL菌液,读取OD600吸光值。
6)将深孔板1500rpm,离心30min。
7)从每孔中取150μL上清液,分别加入50μL氯胺T混匀,避光静置20min后,加入50μL显色剂后,水浴60℃,20min,冷水浴1min,在560nm处测得吸光值,结果见图8。
结果显示,在适合浓度的辅助因子的诱导下,在FeSO4浓度为75mg/L、抗坏血酸浓度为10mg/L和α-酮戊二酸浓度为35mg/L时脯氨酸羟化酶的活性为0.36U/mg,与其它组相比有极显著提高,这也证明了在LB培养基体系中添加一定量的辅助因子,可以使高通量筛选体系更加稳定的筛选出脯氨酸羟化酶的活性。
对Hy593正交实验进行分析如表4所示,显著性分析中抗坏血酸<α-酮戊二酸<FeSO4,说明这三种辅助因子对脯氨酸羟化酶的活性影响顺序为抗坏血酸>α-酮戊二酸>FeSO4。图9分别表明FeSO4、抗坏血酸、α-酮戊二酸在不同浓度下对Hy593酶活性的影响。因为Fe2+有抑菌作用,随着Fe2+离子浓度的升高,大肠杆菌生长受到抑制,所以Fe2+浓度的升高导致Hy593酶活降低。抗坏血酸随着浓度的升高酶活性是先升高后降低,在低浓度时对酶活性的影响较小,在抗坏血酸浓度为10mg/L时,脯氨酸羟化酶活性最高,当浓度大于10mg/L后开始抑制Hy593的酶活。α-酮戊二酸能提高细菌对抗生素的敏感性,所以随着浓度的升高,LB中抗生素浓度未变,大肠杆菌对抗生素的敏感性提高,所以Hy593的活性随着α-酮戊二酸浓度的升高而降低。
表4主旨间效果鉴定
最后应说明的是:以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明实施例技术方案的精神和范围。
Claims (9)
1.一种用于重组胶原修饰的脯氨酸羟化酶筛选方法,其特征在于,其包括如下步骤:
S1:将连接有脯氨酸羟化酶的重组质粒转入宿主菌中,在LB培养基中直接添加脯氨酸和辅助因子共培养发酵,加入IPTG诱导表达,用氯胺T方法检测酶活,高通量筛选出具有酶活的脯氨酸羟化酶;
S2:将连接有步骤S1中筛选出的脯氨酸羟化酶与重组胶原的重组质粒转入宿主菌中,对菌株进行诱导表达,通过Western blot对重组胶原蛋白的羟化修饰作用进行检测。
2.根据权利要求1所述的一种用于重组胶原修饰的脯氨酸羟化酶筛选方法,其特征在于,所述辅助因子选自FeSO4、抗坏血酸和α-酮戊二酸。
3.根据权利要求1所述的一种用于重组胶原修饰的脯氨酸羟化酶筛选方法,其特征在于,所述FeSO4浓度为75mg/L、抗坏血酸浓度为10mg/L和α-酮戊二酸浓度为35mg/L。
4.根据权利要求1所述的一种用于重组胶原修饰的脯氨酸羟化酶筛选方法,其特征在于,所述脯氨酸的量为5g/L。
5.根据权利要求1所述的一种用于重组胶原修饰的脯氨酸羟化酶筛选方法,其特征在于,所述S1中的重组质粒为pACYCDuet-P4H,所述S2中的重组质粒为ACYCDuet-P4H-RC。
6.根据权利要求1所述的一种用于重组胶原修饰的脯氨酸羟化酶筛选方法,其特征在于,所述宿主菌为大肠杆菌BL21。
7.根据权利要求1所述的一种用于重组胶原修饰的脯氨酸羟化酶筛选方法,其特征在于,所述S1的操作如下:
培养含有PACYCDuet-P4H的BL21菌液在OD600处吸光值为0.8-1.0时加入0.5mM IPTG,28℃诱导,诱导24h,诱导完成后,离心取350μL上清液,加入150μL氯胺T溶液,避光静置20min,加入150μL显色剂,水浴60℃,20min,最后冷却至室温,取200μL置于酶标板中,在OD560测得吸光值,计算酶活。
8.根据权利要求1所述的一种用于重组胶原修饰的脯氨酸羟化酶筛选方法,其特征在于,所述S2的操作如下:
对pACYCDuet-1-P4H-RC的BL21(DE3)菌株进行诱导表达,24h取2mL菌液,离心收集菌体,加入300μL 1X蛋白loading Buffer重悬菌体,金属浴95℃,5min后进行Western Blot验证。
9.权利要求1-8任一项所述的筛选方法在筛选用于重组胶原修饰的脯氨酸羟化酶中的应用。
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