CN116855470B - 酰基转移酶MsAcT变体、融合蛋白及其固定方法和应用 - Google Patents
酰基转移酶MsAcT变体、融合蛋白及其固定方法和应用 Download PDFInfo
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Abstract
本发明公开了一种酰基转移酶MsAcT变体，是在野生型酰基转移酶MsAcT的基础上进行S11C、T93A、F150I、F154A、T161S五位点突变。还公开了其融合蛋白、编码基因、表达质粒及宿主菌，以及其固定方法和在催化寡肽乙酰基化或棕榈酰基化中的应用。本发明对野生型酰基转移酶MsAcT改造，获得五位点突变体，并且在突变体的N端插入促溶的Spy‑GST标签，获得的酰基转移酶变体可以生物合成，且酶活明显高于野生型酰基转移酶。本发明还将酰基转移酶变体固定在SpyCather树脂上，获得稳定性更高的固定化酰基转移酶变体，使其更具有工业应用的价值。本发明的固定化酰基转移酶变体可催化寡肽乙酰基化和棕榈酰基化，催化效率高，且不产生有害副产物。
Description
技术领域
本发明涉及一种酰基转移酶MsAcT变体、融合蛋白、编码基因、表达质粒及宿主菌，以及其固定方法和在催化寡肽乙酰基化或棕榈酰基化中的应用，涉及生物合成技术领域。
背景技术
寡肽又称为小肽、低聚肽或称为小分子活性肽，是由2～10个氨基酸彼此缩合形成的化合物，是多肽的一种分类。20世纪60年代Newey和Smyth(1959,1960)第一次提供了肽被完整吸收的资料。蛋白质在小肠中被消化成氨基酸与寡肽，且寡肽可以完整的进入肠粘膜细胞并水解成氨基酸进入血液循环。寡肽具有不需要消化直接吸收、吸收时不消耗能量、不增加肠胃功能负担、100％被人体吸收等特点。寡肽的修饰主要为酰基化修饰，包括乙酰化和棕榈酰化，修饰后的寡肽往往具有活性好、透皮性高和稳定性好等优点，因此在合成寡肽时，往往会引入酰基化修饰来提高寡肽产品的性能。
传统的寡肽合成主要分为化学合成和生物合成。化学合成利用固相或者液相介质，将肽链依照顺序逐个添加到合成中间产物上。这种化学合成的方法适用于二肽或者三肽的合成，但应用在较长寡肽的合成时，会出现反应步骤多，副产物和中间产物多，产量低和提纯困难等问题。生物合成是利用生物系统的蛋白表达技术，将寡肽融合蛋白表达在宿主内部，再经过蛋白纯化和寡肽释放来获得目的寡肽。这种方法的优势是不受寡肽长度和氨基酸序列的影响，可以简单高效地合成寡肽，副产物少，也是目前工业上合成寡肽的重要方法。但是，生物合成的寡肽往往不带有酰基化修饰，因此需在寡肽合成制备后，再进行化学酰基化修饰，这种修饰方法对寡肽的性能和稳定都是极大的挑战，并容易产生非理想的有害副产物。因此，更加绿色高效的酶介导寡肽酰基化修饰方法，也是寡肽生物合成领域的迫切需求。
耻垢分枝杆菌(Mycobacterium smegmatis)的酰基转移酶MsAcT(NCBI SequenceID:WP_011731199.1)是近年来发现的高效酰基转移酶，具有效率高、底物谱广泛、催化条件温和和催化环境适应度高等优点，已经广泛应用在合成生物学领域各类药物中间体和化妆品原料的合成催化过程，是酶催化合成领域的明星蛋白。因此，MsAcT具有催化寡肽酰基化修饰的潜能。
发明内容
本发明公开了一种酰基转移酶MsAcT变体，是在野生型酰基转移酶MsAcT的基础上进行S11C、T93A、F150I、F154A、T161S五位点突变。
为提高其溶解性，在酰基转移酶MsAcT变体的N端加入Spy-GST标签，带有标签的酰基转移酶MsAcT变体融合蛋白的氨基酸序列如SEQ ID NO:1所示，编码基因序列如SEQ IDNO:2所示。
本发明还公开了上述酰基转移酶MsAcT变体融合蛋白的表达质粒，质粒载体为pET22b，酰基转移酶MsAcT变体融合蛋白的编码基因插入到pET22b的PelB信号肽下游。将表达质粒转入到Rosetta(DE3)感受态中，获得表达酰基转移酶MsAcT变体融合蛋白的宿主菌。
为提高酰基转移酶MsAcT变体融合蛋白的稳定性，本发明还公开了上述的酰基转移酶MsAcT变体融合蛋白的固定方法，其步骤包括：
(1)将SpyCather-Cys加入到环氧基树脂中，制得SpyCather树脂；
(2)将上述的宿主菌培养以表达酰基转移酶MsAcT变体融合蛋白，破碎菌体，离心获取上清液，上清液与SpyCather树脂一起孵育，离心，获得固定有酰基转移酶MsAcT变体的SpyCather树脂。
优选的，所述环氧基树脂为是表面含有环氧基团，能与伯胺或者巯基进行共价交联反应的树脂；所述SpyCather-Cys是指含有半胱氨酸残基的SpyCather蛋白。SpyCather-Cys的半胱氨酸残基与环氧基团反应，使得SpyCather蛋白耦合到树脂表面。
本发明对野生型酰基转移酶MsAcT改造，获得S11C、T93A、F150I、F154A、T161S五位点突变体，并且在突变体的N端插入促溶的Spy-GST标签，获得的酰基转移酶变体可以生物合成，且酶活明显高于野生型酰基转移酶。本发明还将酰基转移酶变体固定在SpyCather树脂上，获得稳定性更高的固定化酰基转移酶变体，使其更具有工业应用的价值。本发明的固定化酰基转移酶变体可催化寡肽乙酰基化和棕榈酰基化，催化效率高，且产物纯净，不产生有害副产物。
附图说明
图1为野生型MsAcT表达和纯化结果。
图2为重组MsAcT野生型表达和纯化结果。
图3为重组MsAcT变体表达和纯化结果。
图4为野生型MsAcT与MsAcT变体酶活性比较结果。
图5为MsAcT变体固定化原理图。
图6为MsAcT变体在固定化前后酶活比较结果。
图7为MsAcT变体催化寡肽乙酰基化和棕榈酰基化的化学式。
具体实施方式
以下结合附图，通过实施例进一步说明本发明，但不作为对本发明的限制。以下提供了本发明实施方案中所使用的具体材料及其来源。但是，应当理解的是，这些仅仅是示例性的，并不意图限制本发明，与如下试剂和仪器的类型、型号、品质、性质或功能相同或相似的材料均可以用于实施本发明。下述实施例中所使用的实验方法如无特别说明，均为常规方法。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1
在本实施例中，我们对耻垢分枝杆菌(Mycobacterium smegmatis)的酰基转移酶MsAcT(NCBI Sequence ID:WP_011731199.1)进行酶活和序列同源性改造，使用的DrugDiscovery和Consensus Finder，最终获得S11C、T93A、F150I、F154A、T161S五位点突变体。
我们将获得的野生型和突变体进行了密码子优化，表达在pET28a载体上，发现表达的蛋白为无活性的包涵体(如图1)。为了进一步提高蛋白表达的可溶性，我们在突变体前面插入了促溶的Spy-GST标签，最终获得的酶变体序列见SEQ ID NO:1，核苷酸序列经密码子优化后见SEQ ID NO:2。为了进一步提高蛋白的可溶性，我们将合成的SEQ ID NO:2插入到pET22b的PelB的信号肽下游，使用的酶切位点为NcoI和XhoI双酶切，让表达的融合蛋白通过PelB信号肽分泌到大肠杆菌的周质空间中。野生型的MsAcT做同样的处理。
构建好的质粒转入到全式金生物的Rosetta(DE3)大肠杆菌感受态中，冰浴30min后，42℃热激90s，继续冰浴5min后，涂布到含100mg/L氨苄霉素的LB固体平板上。37℃倒置培养过夜。挑取单克隆于含100mg/L氨苄霉素的LB液体培养基中，200rpm 37℃震荡培养至OD600值达到0.6，加入0.2mM IPTG，25℃200rpm继续培养24h。离心收集菌株，加入10mM PBS(含蛋白酶抑制剂)重悬进行高压破碎后，使用天地人和的HisPur Ni NTA kit和GSTPurGlutathione kit按照其说明书进行蛋白纯化。纯化结果见图1、图2和图3，其中图1为野生型MsAcT的表达纯化结果，图2为融合了Spy-GST标签的野生型MsAcT的表达纯化结果，图3为融合了Spy-GST标签的MsAcT变体的表达纯化结果，融合了Spy-GST标签的MsAcT变体蛋白可溶性表达更好，纯化效果更好。我们随后对MsAcT进行了酰基转移酶活性测试，使用原鑫生物的酰基转移酶(AAT)活性检测试剂盒，酶活结果见图4，将野生型MsAcT的酶活定义为1，可以看出，MsAcT变体的酰基转移酶活性是野生型的2.8倍。
实施例2
在本实施例中，我们对表达的酰基转移酶变体进行了固定化，原理见图5。
双酚A型缩水甘油醚环氧树脂(Sigma Aldrich)经聚合固化后，使用研磨破碎机制成2-3um的树脂颗粒。取1g树脂颗粒，加入10mg SpyCather-Cys(Bio-rad,TZC025)后，在0.5M氯化钠(pH 7.4)的条件下，20℃处理6h。离心获得树脂沉淀，即为制备好的SpyCather树脂。
挑取单克隆于含100mg/L氨苄霉素的LB液体培养基中，200rpm 37℃震荡培养至OD600值达到0.6时，加入0.2mM IPTG，25℃200rpm继续培养24h。离心收集菌株，加入10mMPBS(含蛋白酶抑制剂)重悬进行高压破碎后，取上清，加入质量比0.2％的制备好的树脂，室温震荡孵育2h后，离心收集树脂，PBS清洗树脂3次后备用。使用原鑫生物的酰基转移酶(AAT)活性检测试剂盒检测固定化前后的酰基转移酶变体的活性和稳定性。结果见图6，固定化的酰基转移酶变体仍保留80％的酰基转移酶变体活性，且具有更好的稳定性。
实施例3
在本实施例中，我们利用固定化的酰基转移酶变体对小分子寡肽进行催化测试。
乙酰基化催化(原理图见图7)：10ml体系中加入1ml乙酸乙酯，0.5g的酰基转移酶固定化树脂，0.5g寡肽,100mM磷酸盐缓冲液，pH 8.0。25℃搅拌过夜。HPLC检测乙酰化效率。结果显示，各寡肽乙酰基化比例为二肽-1(71.4％)、四肽-5(52.6％)、四肽-9(57.2％)、六肽-8(35.3％)。
棕榈酰基化催化(原理图见图7)：10ml体系中加入100ul棕榈酸乙酯，500ul叔丁醇，0.5g的酰基转移酶固定化树脂，0.5g寡肽,100mM磷酸盐缓冲液，pH 8.0。25℃搅拌过夜。HPLC检测棕榈酰化效率。结果显示，各寡肽棕榈酰基化比例为三肽-1(17.8％)、三肽-5(12.1％)、三肽-8(9.5％)、四肽-7(13.9％)、四肽-11(8.4％)、五肽-4(12.5％)。
Claims (8)
1.一种酰基转移酶MsAcT变体融合蛋白，其特征在于其氨基酸序列如SEQ ID NO:1所示。
2.权利要求1所述的酰基转移酶MsAcT变体融合蛋白的编码基因，其特征在于其DNA序列如SEQ ID NO:2所示。
3.权利要求1所述的酰基转移酶MsAcT变体融合蛋白的表达质粒。
4.根据权利要求3所述的酰基转移酶MsAcT变体融合蛋白的表达质粒，其特征在于质粒载体为pET22b，酰基转移酶MsAcT变体融合蛋白的编码基因插入到pET22b的PelB信号肽下游。
5.包含权利要求3或4所述的酰基转移酶MsAcT变体融合蛋白的表达质粒的宿主菌。
6.权利要求1所述的酰基转移酶MsAcT变体融合蛋白在催化寡肽乙酰基化或棕榈酰基化中的应用。
7.权利要求1所述的酰基转移酶MsAcT变体融合蛋白的固定方法，其特征在于其步骤包括：
（1）将SpyCather-Cys加入到环氧基树脂中，制得SpyCather树脂；
（2）将权利要求5所述的宿主菌培养以表达酰基转移酶MsAcT变体融合蛋白，破碎菌体，离心获取上清液，上清液与SpyCather树脂一起孵育，离心，获得固定有酰基转移酶MsAcT变体的SpyCather树脂。
8.根据权利要求7所述的固定方法，其特征在于所述环氧基树脂为是表面含有环氧基团，能与伯胺或者巯基进行共价交联反应的树脂；所述SpyCather-Cys是指含有半胱氨酸残基的SpyCather蛋白。
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