CN108823225B - 蛋白质翻译过程中直接实现脂肪酸修饰的表达系统及应用 - Google Patents
蛋白质翻译过程中直接实现脂肪酸修饰的表达系统及应用 Download PDFInfo
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Abstract
本发明公开了一种在翻译过程中直接实现蛋白质脂肪酸修饰的表达系统,包括脂肪酸类非天然氨基酸ε‑N‑庚酰基赖氨酸(ε‑N‑heptanoyl‑Lysine,HepoK)盐酸盐或三氟乙酸盐,以及对HepoK特异的正交氨酰tRNA合成酶突变体和表达宿主。本发明利用遗传编码非天然氨基酸技术(Genetically Encoding Unnatural Amino Acid,GEUAA)将HepoK定点插入目标蛋白中琥珀终止密码子(TAG)编码的位置,在表达宿主体内高效、特异、定量、均一地实现了蛋白质脂肪酸修饰,彻底克服了体外化学修饰效率偏低、修饰均一度差、产物分离困难等多种局限。本发明还首次实现了药物蛋白胰高血糖素样肽‑1(GLP‑1)的共翻译脂肪酸修饰,并证明修饰不影响其生物活性。
Description
技术领域
本发明属于生物制药技术领域,特别涉及一种药物蛋白的制备。
背景技术
随着生物技术的高速发展,蛋白质多肽类药物已成为21世纪医药研发领域中最活跃、进展最快的药物之一。目前被批准上市的蛋白药物超过200种,除了抗体和Fc融合蛋白以外,还有很大一部分低分子量蛋白药物,例如激素、生长因子、细胞因子、凝血因子等,由于相对较小的分子尺寸,它们容易被肾过滤(阈值为~50kDa)从血循环中迅速清除,加上蛋白酶降解和受体介导的胞吞作用,使其血液半衰期普遍偏短(几分钟至几小时)。为了维持药物作用的有效浓度,给药频率和剂量必须加大,潜在副作用风险增加,同时也导致患者的依从性大幅度降低。为了克服上述问题,研究人员逐渐发展出了多种蛋白长效化技术,通过提高蛋白分子的稳定性(例如定点突变)、增加流体动力学体积(例如聚合缓释、糖修饰、PEG修饰)或者利用新生儿Fc受体(FcRn)介导的再循环(例如白蛋白融合、Fc融合)等来延长蛋白药物体内的半衰期。然而现有技术均存在各种限制而缺乏通用性。蛋白药物的长效化机制和技术研究仍然是生物制药的重要方向。
长期以来,与人血清白蛋白(HSA)融合或结合被认为是延长药物蛋白半衰期的优选策略。因为HSA在人体内具有非免疫原性,生物相容性和可降解性;并且HSA分子尺寸超过肾过滤阈值,还可利用FcRn介导的细胞内降解逃逸机制,使得HSA在人体内血清半衰期长达19天。人们期望通过药物蛋白与HSA的融合表达来实现药物蛋白的长效化。然而,大部分结果难以达到预期,因为晶体结构显示,FcRn与HSA结合时,会同时与其N端的D1亚基以及C端的D3亚基相互作用。如果将蛋白直接融合到HSA上,可能会遮蔽这两个亚基中的一个,降低药物蛋白-HSA偶联物与FcRn的亲和力,从而不能有效延长其半衰期,而且与HSA的融合还可能阻碍药物蛋白与靶点的结合,从而降低药效。因此脂肪酸介导的、与HSA可逆地结合是个更有效的长效化方案:首先,HSA具有多达7个以上的脂肪酸特异结合位点(见图1);其次,脂肪酸修饰有更多的位置选择,除了药物蛋白的N端,C端,理论上其他氨基酸残基的官能团也可进行脂肪酸修饰;再次,脂肪酸分子尺寸小,对药物蛋白自身空间结构影响小,药物可以保持更加良好的生物活性,而且脂肪酸与HSA结合后,几乎不影响其与FcRn的结合;最后,脂肪酸是构成人体脂肪、类脂和细胞膜磷脂的重要成分,脂肪酸修饰还有助于提高药物的脂溶性,增大肠道黏膜透过性及吸收效率,增强药物与受体的结合,甚至有潜能实现蛋白药物口服给药。因此蛋白质的脂肪酸修饰是新一代的蛋白药物长效化方案。
天然蛋白的脂肪酸修饰(脂化)主要形式有N-肉豆蔻酰化、S-法尼基化、S-棕榈酰化、S-香叶基香叶基化,胆固醇修饰以及磷脂酰乙醇胺修饰等,均在体内通过各种酶来催化实现,具有底物特异性和特定的修饰位点。为了降低免疫原性,蛋白药物脂肪酸修饰原则上模拟天然脂质蛋白结构,但蛋白药物自身通常并非脂化酶的底物,也无脂化位点,因而无法通过普通的重组蛋白生产体系或者体外酶催化来获取。迄今为止,蛋白药物的脂肪酸修饰主要通过化学全合成(多肽)或者体外化学偶联来实现(蛋白)。已经上市的脂肪酸修饰药物如地特胰岛素、德谷胰岛素、利拉鲁肽及索马鲁肽,均通过体外化学反应来生产:地特胰岛素采用肉豆蔻酸琥珀酰亚胺脂酰化第29位赖氨酸残基来完成修饰,其他三种则是以谷氨酸作为接头连接脂肪酸链和药物分子。
现有的蛋白质体外化学脂肪酸修饰技术存在诸多问题和挑战:1.修饰选择性不高。现有化学合成法利用的主要是蛋白的N端氨基、C端羧基、半胱氨酸上的巯基、丝氨酸或苏氨酸上的羟基等官能团。除了部分小肽(例如胰高血糖素样肽-1,GLP-1)能提供单一的修饰位点外,大部分蛋白具有多个化学性质类似的氨基酸残基,无法进行高选择性的定点修饰。2.产物不均一。由于蛋白质化学反应区域选择性不高,多位点修饰更是容易造成各个位点的修饰程度不一,形成混合物,从而造成产品不均一,需要复杂的后处理来分离纯化。3.修饰后蛋白可能失活。由于修饰位点无法选择,随机修饰位点可能处于蛋白的生物活性中心或别构效应位点,从而造成蛋白失活。4.生产工艺复杂。蛋白由20种氨基酸组成,侧链含有氨基、巯基、羧基、羟基等多种化学活性基团,进行修饰反应的时候,需要对敏感基团进行预保护和脱保护,额外增加工艺步骤,产率与质量也随之下降。5.反应条件限制苛刻。蛋白活性依赖于三、四级结构,而高级结构对温度、压力、有机溶剂以及金属离子等耐受性不高。因此在有机化学中可以高效进行的反应(例如酰胺化)用于脂肪酸加成的时候,效率通常不高。如果使用的反应条件强烈,容易造成蛋白氧化、旋光异构、降解、聚合,从而引起蛋白损伤和变性。
发明内容
为了从根本上解决蛋白质翻译后再用化学方法进行脂肪酸修饰带来的各种问题,本发明用生物合成的方法在翻译过程中直接实现脂肪酸修饰。具体而言,本发明设计并合成脂肪酸类非天然氨基酸ε-N-庚酰基赖氨酸(ε-N-heptanoyl-Lysine,HepoK)的盐酸盐或者三氟乙酸盐,然后定向进化正交氨酰tRNA-合成酶系统,使之对HepoK特异,利用遗传编码非天然氨基酸技术(Genetically Encoding Unnatural Amino Acid,GEUAA)将HepoK定点插入目标蛋白中琥珀终止密码子(TAG)编码的位置,在表达宿主体内高效、特异、定量、均一地实现蛋白质脂肪酸修饰(见图2)。
本发明第一个目的是提供一种在翻译过程中直接实现脂肪酸修饰的表达系统,包括:
(1)脂肪酸链非天然氨基酸:ε-N-庚酰基赖氨酸(HepoK)的盐酸盐或三氟乙酸盐,其分子结构式如下:
ε-N-庚酰基赖氨酸(HepoK)盐酸盐(ε-N-heptanoyl-Lysine HCl salt or(S)-1-carboxy-5-heptanamidopentan-1-aminium chloride),化学式:C13H27ClN2O3,分子量294.82;或ε-N-庚酰基赖氨酸(HepoK)三氟乙酸盐(ε-N-heptanoyl-LysineTFA salt or(S)-1-carboxy-5-heptanamidopentan-1-aminium 2,2,2-trifluoroacetate),化学式:C15H27F3N2O5,分子量372.38。
(2)HepoK特异的正交氨酰tRNA合成酶;
(3)表达宿主。
进一步地,所述正交氨酰tRNA合成酶为突变合成酶1,其氨基酸序列为:
MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMMAPNLANYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFFQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL。
突变合成酶1的核苷酸序列为:
ATGGATAAAAAGCCTTTGAACACTCTGATTTCTGCGACCGGTCTGTGGATGTCCCGCACCGGCACCATCCACAAAATCAAACACCATGAAGTTAGCCGTTCCAAAATCTACATTGAAATGGCTTGCGGCGATCACCTGGTTGTCAACAACTCCCGTTCTTCTCGTACCGCTCGCGCACTGCGCCACCACAAATATCGCAAAACCTGCAAACGTTGCCGTGTTAGCGATGAGGACCTGAACAAATTCCTGACCAAAGCTAACGAGGATCAGACCTCCGTAAAAGTGAAGGTAGTAAGCGCTCCGACCCGTACTAAAAAGGCTATGCCAAAAAGCGTGGCCCGTGCCCCGAAACCTCTGGAAAACACCGAGGCGGCTCAGGCTCAACCATCCGGTTCTAAATTTTCTCCGGCGATCCCAGTGTCCACCCAAGAATCTGTTTCCGTACCAGCAAGCGTGTCTACCAGCATTAGCAGCATTTCTACCGGTGCTACCGCTTCTGCGCTGGTAAAAGGTAACACTAACCCGATTACTAGCATGTCTGCACCGGTACAGGCAAGCGCCCCAGCTCTGACTAAATCCCAGACGGACCGTCTGGAGGTGCTGCTGAACCCAAAGGATGAAATCTCTCTGAACAGCGGCAAGCCTTTCCGTGAGCTGGAAAGCGAGCTGCTGTCTCGTCGTAAAAAGGATCTGCAACAGATCTACGCTGAGGAACGCGAGAACTATCTGGGTAAGCTGGAGCGCGAAATTACTCGCTTCTTCGTGGATCGCGGTTTCCTGGAGATCAAATCTCCGATTCTGATTCCGCTGGAATACATTGAACGTATGGGCATCGATAATGATACCGAACTGTCTAAACAGATCTTCCGTGTGGATAAAAACTTCTGTCTGCGTCCGATGATGGCGCCGAACCTTGCTAACTATGCTCGTAAACTGGACCGTGCCCTGCCGGACCCGATCAAAATTTTCGAGATCGGTCCTTGCTACCGTAAAGAGTCCGACGGTAAAGAGCACCTGGAAGAATTCACCATGCTGAACTTCTTCCAGATGGGTAGCGGTTGCACGCGTGAAAACCTGGAATCCATTATCACCGACTTCCTGAATCACCTGGGTATCGATTTCAAAATTGTTGGTGACAGCTGTATGGTGTATGGCGATACGCTGGATGTTATGCACGGCGATCTGGAGCTGTCTTCCGCAGTAGTGGGCCCAATCCCGCTGGATCGTGAGTGGGGTATCGACAAACCTTGGATCGGTGCGGGTTTTGGTCTGGAGCGTCTGCTGAAAGTAAAACACGACTTCAAGAACATCAAACGTGCTGCACGTTCCGAGTCCTATTACAATGGTATTTCTACTAACCTGTAA。
所述正交氨酰tRNA合成酶还可以为突变合成酶2,其氨基酸序列为:
MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMMSPNIANYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFFQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL。
突变合成酶2的核苷酸序列为:
ATGGATAAAAAGCCTTTGAACACTCTGATTTCTGCGACCGGTCTGTGGATGTCCCGCACCGGCACCATCCACAAAATCAAACACCATGAAGTTAGCCGTTCCAAAATCTACATTGAAATGGCTTGCGGCGATCACCTGGTTGTCAACAACTCCCGTTCTTCTCGTACCGCTCGCGCACTGCGCCACCACAAATATCGCAAAACCTGCAAACGTTGCCGTGTTAGCGATGAGGACCTGAACAAATTCCTGACCAAAGCTAACGAGGATCAGACCTCCGTAAAAGTGAAGGTAGTAAGCGCTCCGACCCGTACTAAAAAGGCTATGCCAAAAAGCGTGGCCCGTGCCCCGAAACCTCTGGAAAACACCGAGGCGGCTCAGGCTCAACCATCCGGTTCTAAATTTTCTCCGGCGATCCCAGTGTCCACCCAAGAATCTGTTTCCGTACCAGCAAGCGTGTCTACCAGCATTAGCAGCATTTCTACCGGTGCTACCGCTTCTGCGCTGGTAAAAGGTAACACTAACCCGATTACTAGCATGTCTGCACCGGTACAGGCAAGCGCCCCAGCTCTGACTAAATCCCAGACGGACCGTCTGGAGGTGCTGCTGAACCCAAAGGATGAAATCTCTCTGAACAGCGGCAAGCCTTTCCGTGAGCTGGAAAGCGAGCTGCTGTCTCGTCGTAAAAAGGATCTGCAACAGATCTACGCTGAGGAACGCGAGAACTATCTGGGTAAGCTGGAGCGCGAAATTACTCGCTTCTTCGTGGATCGCGGTTTCCTGGAGATCAAATCTCCGATTCTGATTCCGCTGGAATACATTGAACGTATGGGCATCGATAATGATACCGAACTGTCTAAACAGATCTTCCGTGTGGATAAAAACTTCTGTCTGCGTCCGATGATGTCGCCGAACATTGCTAACTATGCGCGTAAACTGGACCGTGCCCTGCCGGACCCGATCAAAATTTTCGAGATCGGTCCTTGCTACCGTAAAGAGTCCGACGGTAAAGAGCACCTGGAAGAATTCACCATGCTGAACTTCTTCCAGATGGGTAGCGGTTGCACGCGTGAAAACCTGGAATCCATTATCACCGACTTCCTGAATCACCTGGGTATCGATTTCAAAATTGTTGGTGACAGCTGTATGGTGTATGGCGATACGCTGGATGTTATGCACGGCGATCTGGAGCTGTCTTCCGCAGTAGTGGGCCCAATCCCGCTGGATCGTGAGTGGGGTATCGACAAACCTTGGATCGGTGCGGGTTTTGGTCTGGAGCGTCTGCTGAAAGTAAAACACGACTTCAAGAACATCAAACGTGCTGCACGTTCCGAGTCCTATTACAATGGTATTTCTACTAACCTGTAA。
优选地,所述表达宿主为大肠杆菌、酵母或哺乳动物细胞。
本发明第二个目的是提供上述表达系统在定点修饰蛋白质中的应用。
本发明第三个目的是提供上述表达系统在制备药物蛋白中的应用,特别是在制备人胰高血糖素样肽-1(GLP-1)中的应用。
本发明最后一个目的是提供了一种药物蛋白的制备方法,其特征在于包括以下步骤:
(1)合成脂肪酸链非天然氨基酸-HepoK的盐酸盐或三氟乙酸盐,分子结构式如下:
(2)将蛋白基因特定位点DNA编码突变为TAG,获得药物蛋白表达载体;
(3)将药物蛋白表达载体与HepoK特异的正交氨酰tRNA合成酶及正交tRNA表达载体共转,在大肠杆菌、酵母或哺乳动物细胞表达系统中制备带HepoK的药物蛋白。
进一步地,药物蛋白为人胰高血糖素样肽-1(GLP-1),HepoK定点取代第26位赖氨酸。
遗传编码HepoK实现了蛋白质共翻译的(co-translational)脂肪酸修饰,从原理上革新地规避现有技术中蛋白翻译后(post-translational)再化学修饰的各种缺陷。具有以下优点:
(1)蛋白无需经历体外化学反应。连接脂肪酸的不再是功能蛋白,而是单一的氨基酸分子。因此可以按化学反应需要使用温度、金属离子、有机试剂及分离手段,无需考虑其他氨基酸侧链基团的干扰,也不用担心化学反应使蛋白质变性或功能受损。
(2)修饰位点的选择高度特异、灵活可控。GEUAA技术结合了原理清晰、操控性强的重组蛋白生产技术(从DNA到蛋白质)和体内酶催化底物专一性特点。修饰位点的选择通过DNA点突变方法将TAG密码子引入到目标基因的任意特定位置来实现,而HepoK将被正交的tRNA-合成酶按蛋白翻译原则定点放置在由TAG密码子指定的特异位点,最终呈现出定点、均一的脂肪酸修饰产物。
(3)修饰效率高、产物均一。与化学修饰低反应效率形成鲜明对比的是,遗传编码引入的HepoK是以近100%的效率定量引入蛋白质中的,所以产物均一,分离纯化简单。
(4)易于保全蛋白活性。GEUAA技术原理上可将HepoK放置在蛋白任意位置,通过简单的DNA突变即可实现全蛋白序列筛选,避开活性和别构调控区域,从而保全蛋白功能。
本发明通过合成生物方法改造生物体蛋白质合成机制,在共翻译过程中把脂肪酸修饰直接、定点、定量地引入目标蛋白质。此方法在表达宿主体内温和、均一、高效地实现蛋白质脂肪酸修饰,从原理上彻底克服了现有体外化学修饰效率偏低、产物分离困难、修饰均一度差等多种局限。本发明首次实现无需体外化学反应的纯生物制备来一步到位地制备脂肪酸修饰的蛋白药物。
本发明在蛋白质脂肪酸修饰技术上的突破允许我们系统深入地研究脂肪酸修饰蛋白的理化、生物学性能以及体内动力学性质,为药物蛋白长效化提供理论基础,并有望发展出新一代的蛋白长效化平台技术。
附图说明
图1是与不同链长脂肪酸复合的HSA的结构示意图。结合的脂肪酸以球形结构显示。数字1-9表示不同的脂肪酸结合位点。Cn:0表示含n个碳原子的脂肪酸,不饱和键为0。
图2是遗传编码带脂肪酸侧链的氨基酸以一步实现药物蛋白的定点与定量脂肪酸修饰示意图。
图3是本发明设计线路图。
图4是ε-N-庚酰基赖氨酸(HepoK)盐酸盐的氢谱和碳谱图
图5是吡咯酪氨酸(Pyl)及其氨酰tRNA合成酶(PylRS)复合物的晶体结构图(PDB序号2ZCE),数字标记的是饱和突变的活性中心氨基酸残基。
图6是定向进化氨酰tRNA合成酶示意图。
图7是HepoK特异的正交氨酰tRNA合成酶(PylRS)突变体的筛选结果图。其中图A和C是不加HepoK的培养盘,图B和D是加1mM HepoK的培养盘。方框标记出的第13号和第21号菌株,呈现HepoK依赖性的生长和绿色荧光,说明此菌中含有对HepoK特异的PylRS突变体。
图8是突变合成酶1的氨基酸序列与野生型序列的对比图。
图9是突变合成酶2的氨基酸序列与野生型序列的对比图。
图10是胰高血糖素样肽-1(GLP-1)结构示意图。
图11定点插入HepoK的肌红蛋白的western结果图。
图12定点插入HepoK的肌红蛋白的质谱结果图。
图13是ForteBio测量HSA与脂肪酸修饰蛋白之间亲和力的示意图。
图14HepoK引入前后GLP-1与HSA亲和力的ForteBio测试结果图。
图15胰高血糖素样肽-1(GLP-1)活性分析法示意图
图16HepoK引入前后GLP-1生物活性测量结果图。
具体实施方式
借助于下述具体实施方式可更好地理解本发明,然而,这些具体实施方式仅用于举例说明本发明,不应被解释为对本发明的限制。同样的原理和方法可以用来合成和遗传编码不同长度的脂肪酸类非天然氨基酸,用于同样的目的。
实施例1:化学合成含HepoK的盐酸盐或三氟乙酸盐
α-N-Boc-赖氨酸在碱性条件先与庚酰氯反应生成ε-N-庚酰-α-N-Boc-赖氨酸,在酸性条件下(TFA or HCl)脱除Boc保护基,制备相应的ε-N-庚酰-赖氨酸三氟乙酸盐或盐酸盐,并用核磁共振(NMR)确认(见图4)。
实施例2:氨酰tRNA合成酶库的构建
遗传编码非天然氨基酸技术(GEUAA)是在活细胞中利用正交tRNA/正交的氨酰tRNA-合成酶分子对来识别琥珀终止密码子(TAG),通过核糖体在蛋白质中定点插入非天然氨基酸的方法,而吡咯赖氨酸正是自然界使用该原理的代表。因此本发明以Methanosarcina mazei吡咯赖氨酸(Pyl)的氨酰tRNA合成酶(PylRS)为模板,根据已经发表的晶体结构,采用简并引物突变法,对其氨基酸结合位点中的Leu301,Ala302,Leu305,Tyr306,Leu309,Cys348等位置进行饱和突变,构建>108库容的氨酰tRNA合成酶库(见图5)。
实施例3:氨酰tRNA合成酶的筛选
应用正向和反向双选择方法筛选以上氨酰tRNA合成酶库,
1)正向筛选:将以上合成酶库质粒与含有琥珀终止密码子(TAG)的氯霉素抗性基因及含有TAG的绿色荧光蛋白基因的质粒共转染大肠杆菌DH10β电感受态,并在含有氯霉素和HepoK盐酸盐或三氟乙酸盐(1mM)的培养基培养。如果PylRS识别天然氨基酸或者HepoK,则可读通TAG,则氯霉素抗性基因及绿色荧光蛋白表达,大肠杆菌存活并发绿色荧光。
2)反向筛选:提取存活大肠杆菌中的PylRS质粒,与含有琥珀终止密码子(TAG)的barnase毒蛋白基因的质粒共转染大肠杆菌DH10β电感受态,在不含HepoK盐酸盐或三氟乙酸盐的培养基中培养。如PylRS识别天然氨基酸,则读通barnase毒蛋白基因,大肠杆菌死亡。存活细菌所含PylRS即是专一性识别HepoK的PylRS突变体。
3)再次提取存活细菌中表达PylRS的质粒,利用上述方法,进行多轮高通量正反双筛选,定向进化,最终筛选得到特异性识别HepoK的高效氨酰tRNA合成酶。
定向进化正交的氨酰tRNA合成酶(PylRS),使之能够把HepoK特异、定点地插入到琥珀终止密码子(TAG)编码的位置(具体路线见图6)。图中CAT是氯霉素乙酰转移酶,全长表达的CAT能使细菌在氯霉素中存活;GFP是绿色荧光蛋白,全长表达的GFP能使得细菌在紫外光照下发出绿色荧光;Barnase是RNA降解酶,全长表达的barnase将杀死细菌。正向选择选出能装载天然氨基酸和HepoK的PylRS突变体,而反向选择去除那些装载天然氨基酸的PylRS突变体,因此最终存活的菌株就含有专一性装载HepoK的PylRS突变体。
通过以上筛选方法,得到对HepoK特异的氨酰tRNA合成酶突变体,如图7所示,分别为突变合成酶1和突变合成酶2。
(1)突变合成酶1
氨基酸序列:
MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMMAPNLANYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFFQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL。
核苷酸序列:
ATGGATAAAAAGCCTTTGAACACTCTGATTTCTGCGACCGGTCTGTGGATGTCCCGCACCGGCACCATCCACAAAATCAAACACCATGAAGTTAGCCGTTCCAAAATCTACATTGAAATGGCTTGCGGCGATCACCTGGTTGTCAACAACTCCCGTTCTTCTCGTACCGCTCGCGCACTGCGCCACCACAAATATCGCAAAACCTGCAAACGTTGCCGTGTTAGCGATGAGGACCTGAACAAATTCCTGACCAAAGCTAACGAGGATCAGACCTCCGTAAAAGTGAAGGTAGTAAGCGCTCCGACCCGTACTAAAAAGGCTATGCCAAAAAGCGTGGCCCGTGCCCCGAAACCTCTGGAAAACACCGAGGCGGCTCAGGCTCAACCATCCGGTTCTAAATTTTCTCCGGCGATCCCAGTGTCCACCCAAGAATCTGTTTCCGTACCAGCAAGCGTGTCTACCAGCATTAGCAGCATTTCTACCGGTGCTACCGCTTCTGCGCTGGTAAAAGGTAACACTAACCCGATTACTAGCATGTCTGCACCGGTACAGGCAAGCGCCCCAGCTCTGACTAAATCCCAGACGGACCGTCTGGAGGTGCTGCTGAACCCAAAGGATGAAATCTCTCTGAACAGCGGCAAGCCTTTCCGTGAGCTGGAAAGCGAGCTGCTGTCTCGTCGTAAAAAGGATCTGCAACAGATCTACGCTGAGGAACGCGAGAACTATCTGGGTAAGCTGGAGCGCGAAATTACTCGCTTCTTCGTGGATCGCGGTTTCCTGGAGATCAAATCTCCGATTCTGATTCCGCTGGAATACATTGAACGTATGGGCATCGATAATGATACCGAACTGTCTAAACAGATCTTCCGTGTGGATAAAAACTTCTGTCTGCGTCCGATGATGGCGCCGAACCTTGCTAACTATGCTCGTAAACTGGACCGTGCCCTGCCGGACCCGATCAAAATTTTCGAGATCGGTCCTTGCTACCGTAAAGAGTCCGACGGTAAAGAGCACCTGGAAGAATTCACCATGCTGAACTTCTTCCAGATGGGTAGCGGTTGCACGCGTGAAAACCTGGAATCCATTATCACCGACTTCCTGAATCACCTGGGTATCGATTTCAAAATTGTTGGTGACAGCTGTATGGTGTATGGCGATACGCTGGATGTTATGCACGGCGATCTGGAGCTGTCTTCCGCAGTAGTGGGCCCAATCCCGCTGGATCGTGAGTGGGGTATCGACAAACCTTGGATCGGTGCGGGTTTTGGTCTGGAGCGTCTGCTGAAAGTAAAACACGACTTCAAGAACATCAAACGTGCTGCACGTTCCGAGTCCTATTACAATGGTATTTCTACTAACCTGTAA。
突变合成酶1的氨基酸序列与野生型序列的对比:L301M,Y306A,L309A,C348F,对比图如图8所示。
(2)突变合成酶2
氨基酸序列:
MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMMSPNIANYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFFQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL。
核苷酸序列如下:
ATGGATAAAAAGCCTTTGAACACTCTGATTTCTGCGACCGGTCTGTGGATGTCCCGCACCGGCACCATCCACAAAATCAAACACCATGAAGTTAGCCGTTCCAAAATCTACATTGAAATGGCTTGCGGCGATCACCTGGTTGTCAACAACTCCCGTTCTTCTCGTACCGCTCGCGCACTGCGCCACCACAAATATCGCAAAACCTGCAAACGTTGCCGTGTTAGCGATGAGGACCTGAACAAATTCCTGACCAAAGCTAACGAGGATCAGACCTCCGTAAAAGTGAAGGTAGTAAGCGCTCCGACCCGTACTAAAAAGGCTATGCCAAAAAGCGTGGCCCGTGCCCCGAAACCTCTGGAAAACACCGAGGCGGCTCAGGCTCAACCATCCGGTTCTAAATTTTCTCCGGCGATCCCAGTGTCCACCCAAGAATCTGTTTCCGTACCAGCAAGCGTGTCTACCAGCATTAGCAGCATTTCTACCGGTGCTACCGCTTCTGCGCTGGTAAAAGGTAACACTAACCCGATTACTAGCATGTCTGCACCGGTACAGGCAAGCGCCCCAGCTCTGACTAAATCCCAGACGGACCGTCTGGAGGTGCTGCTGAACCCAAAGGATGAAATCTCTCTGAACAGCGGCAAGCCTTTCCGTGAGCTGGAAAGCGAGCTGCTGTCTCGTCGTAAAAAGGATCTGCAACAGATCTACGCTGAGGAACGCGAGAACTATCTGGGTAAGCTGGAGCGCGAAATTACTCGCTTCTTCGTGGATCGCGGTTTCCTGGAGATCAAATCTCCGATTCTGATTCCGCTGGAATACATTGAACGTATGGGCATCGATAATGATACCGAACTGTCTAAACAGATCTTCCGTGTGGATAAAAACTTCTGTCTGCGTCCGATGATGTCGCCGAACATTGCTAACTATGCGCGTAAACTGGACCGTGCCCTGCCGGACCCGATCAAAATTTTCGAGATCGGTCCTTGCTACCGTAAAGAGTCCGACGGTAAAGAGCACCTGGAAGAATTCACCATGCTGAACTTCTTCCAGATGGGTAGCGGTTGCACGCGTGAAAACCTGGAATCCATTATCACCGACTTCCTGAATCACCTGGGTATCGATTTCAAAATTGTTGGTGACAGCTGTATGGTGTATGGCGATACGCTGGATGTTATGCACGGCGATCTGGAGCTGTCTTCCGCAGTAGTGGGCCCAATCCCGCTGGATCGTGAGTGGGGTATCGACAAACCTTGGATCGGTGCGGGTTTTGGTCTGGAGCGTCTGCTGAAAGTAAAACACGACTTCAAGAACATCAAACGTGCTGCACGTTCCGAGTCCTATTACAATGGTATTTCTACTAACCTGTAA。
突变合成酶2氨基酸序列与野生型序列的对比:L301M,A302S,L305I,Y306A,L309A,C348F,对比图如图9所示。
实施例4:脂肪酸修饰重组蛋白的表达纯化及质谱鉴定
实施例3中得到的HepoK特异的氨酰tRNA合成酶可以在细菌、酵母和哺乳动物细胞中遗传编码HepoK。为方便蛋白表达和质谱检测,首先用模式蛋白Myoglobin(肌红蛋白)来插入待测HepoK,并用全长蛋白质谱和质谱氨基酸测序来分析HepoK插入蛋白的忠信度。药物蛋白则选用人胰高血糖素样肽-1(GLP-1)来测试。GLP-1是一种内源性肠促胰岛素激素,能够促进胰腺β细胞葡萄糖浓度依赖性地分泌胰岛素,是成人Ⅱ型糖尿病药物。然而,天然GLP-1血浆半衰期不足2分钟。本发明将通过遗传编码HepoK对其进行共翻译的脂肪酸修饰,实现体内半衰期的延长。
本发明选择Myoglobin的S4及GLP-1的K26进行脂肪酸修饰(见图10)。
将以上模式蛋白或药物蛋白表达载体与对HepoK特异的氨酰tRNA合成酶共转,在大肠杆菌、酵母或哺乳动物细胞表达系统中制备带HepoK的模式蛋白及药物蛋白。柱层析纯化,并用全长蛋白质质谱和质谱氨基酸测序分析确证HepoK的插入。
具体步骤如下:
1)构建Myoglobin或GLP1蛋白表达质粒于pET28b质粒中,C端带His6标签以利于亲和纯化。
2)将以上质粒进行PCR点突变,构建带有TAG的Myoglobin或GLP1突变蛋白表达质粒。
3)将对HepoK特异的氨酰tRNA合成酶质粒与Myoglobin或GLP1表达质粒共转大肠杆菌BL21(DE3)或者C321ΔA(DE3)(Release factor 1基因敲除菌株,可以提高非天然氨基酸插入效率并有助于多位点插入)。分别挑选3个以上克隆在含有HepoK盐酸盐或三氟乙酸盐的LB培养基中培养,待OD600达到0.5左右的时候,加入诱导剂IPTG及阿拉伯糖。诱导5小时以上,离心收集菌体,超声破碎,取上清,PAGE胶/Western blot分析表达情况。
4)扩大培养,AKTA亲和柱层析纯化。
5)将纯后的蛋白进行质谱分析以确证HepoK的插入和评估插入的忠信度。
利用GEUAA技术重组表达带his6标签的肌红蛋白(Myoglobin),用识别His6标签的抗体进行Western blot检测细菌裂解液(图11)。在加入HepoK盐酸盐或三氟乙酸盐时,肌红蛋白Myoglobin有明显表达(产率8.4mg/L),而不加HepoK盐酸盐或三氟乙酸盐时蛋白翻译终止在引入的TAG位点,无全长的肌红蛋白表达,这说明筛选得到的PylRS突变体能够高效特异地插入脂肪酸类非天然氨基酸HepoK,而不插入天然的氨基酸。
将纯化后的含HepoK的肌红蛋白(Myo-HepoK)进行质谱分析(图12),A)全长Myo-HepoK的质谱分析结果。理论分子量为18449.0Da,测量值为18449.5Da。B)Myo-HepoK的质谱测序结果。肌红蛋白经胰蛋白酶降解为肽段,然后用二级质谱测序。肌红蛋白第4位氨基酸密码子突变为TAG,因此氨基酸序列是TSVUEGEWQLVLHVWAK(U代表HepoK)。质谱测序得到的一系列b和y离子准确且无歧义地显示TAG编码的第4位插入的是HepoK。因此,两种质谱测量结果完全确证HepoK被高忠信度地编码到了肌红蛋白中。
实施例5:脂肪酸修饰蛋白与HSA结合测试及分析
采用生物膜干涉技术(Bio-layer interferometry,BLI)来测量实施例4中得到的含HepoK的胰高血糖素样肽-1(GLP1-Hepok)与HSA的结合。BLI利用光的干涉原理测量分子间的相互作用,包括亲和力。具体测量仪器是RED96,其亲和力测量范围为1mM-10pM,满足脂肪酸修饰蛋白与HSA的亲和力(~10-100uM)测试要求。
将生物素标记的HSA通过链亲和素固定在探针上,测试HSA与GLP1-Hepok的结合与解离(图13),通过ForteBio自带的软件计算亲和力相关数值(Kon,Koff与KD)。具体操作如下:
1)使用Thermo Fisher的生物素标记试剂盒EZ-LinkTM NHS-LC-LC-Biotin,标记HSA(Abcam)。
2)使用Pall公司的超级链亲和素生物传感器固定生物素化的HSA。
3)将GLP1-HepoK稀释成不同的梯度浓度(3.9,7.9,15.8,31.5和63uM)。
4)用Octet RED96测试不同浓度GLP1-HepoK与HSA的结合。
5)计算Kon,Koff与KD。
将3.9,7.9,15.8,31.5和63uM插入HepoK的GLP1(GLP1-HepoK)及野生型的GLP1(GLP1-wt)与固定在sensor上的HSA进行结合与解离实验。结果显示经HepoK修饰的GLP1与HSA的结合(KD=2.89x10-5)明显强于野生型GLP1(图14)。
实施例6:脂肪酸修饰蛋白的生物活性检测
采用细胞内3’,5’-环腺苷酸(cAMP)依赖型蛋白激酶A活性分析法(见图15)测量实施例4中得到的含HepoK的胰高血糖素样肽-1(GLP1-Hepok)的生物学活性。GLP-1与大鼠胰岛瘤细胞RIN-m5F细胞膜上GLP-1受体(G蛋白偶联受体)相互作用后,细胞内腺苷酸环化酶(adenylate cyclase)被激活,导致胞内cAMP水平升高。使用试剂盒裂解细胞,释放出胞内cAMP,cAMP激活蛋白激酶A,蛋白激酶A催化底物磷酸化,消耗ATP,ATP的减少反映为化学发光信号的减弱。具体操作如下:
1)将生长状态良好的大鼠胰岛瘤细胞RIN-m5F接种在96孔板中,7000个/孔,37℃、5%二氧化碳培养箱中静置培养3天。
2)用PBS(pH 8.2)将GLP1-HepoK及野生型的GLP1(GLP1-wt)分别稀释成375、175.5、93.8、46.9、23.4、11.7、5.9、2.9、1.5、0.7ng/mL。
3)移除96孔板中RIN-m5F的培养基,分别加入步骤2中不同浓度的GLP1-HepoK或GLP1-wt,37℃放置5分钟。
4)使用Promega公司的cAMP-GloTMMax Assay试剂盒处理细胞样本,用多功能酶标仪读取相对化学发光单位(RLU)
5)以药物浓度为横坐标,以相对化学发光单位为纵坐标作图,用四参数方程Y=Bottom+(Top-Bottom)/(1+10^((LogEC50-X)*HillSlope))拟合,得到剂量效应曲线,计算出GLP1-HepoK及GLP1-wt的半效浓度。
GLP-1对受体的刺激越强,cAMP水平越高,化学发光信号越弱,结果如图16所示。经过计算得出GLP1-HepoK及GLP1-wt的半效浓度分别为9.5ng/mL和10.0ng/mL,说明GLP1-HepoK与GLP-1受体的结合及功效与野生型GLP-1相当,脂肪酸修饰的GLP-1保持了其原有生物活性。
序列表
<110> 中国科学院理化技术研究所杭州研究院
<120> 蛋白质翻译过程中直接实现脂肪酸修饰的表达系统及应用
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gcttgcggcg atcacctggt tgtcaacaac tcccgttctt ctcgtaccgc tcgcgcactg 180
cgccaccaca aatatcgcaa aacctgcaaa cgttgccgtg ttagcgatga ggacctgaac 240
aaattcctga ccaaagctaa cgaggatcag acctccgtaa aagtgaaggt agtaagcgct 300
ccgacccgta ctaaaaaggc tatgccaaaa agcgtggccc gtgccccgaa acctctggaa 360
aacaccgagg cggctcaggc tcaaccatcc ggttctaaat tttctccggc gatcccagtg 420
tccacccaag aatctgtttc cgtaccagca agcgtgtcta ccagcattag cagcatttct 480
accggtgcta ccgcttctgc gctggtaaaa ggtaacacta acccgattac tagcatgtct 540
gcaccggtac aggcaagcgc cccagctctg actaaatccc agacggaccg tctggaggtg 600
ctgctgaacc caaaggatga aatctctctg aacagcggca agcctttccg tgagctggaa 660
agcgagctgc tgtctcgtcg taaaaaggat ctgcaacaga tctacgctga ggaacgcgag 720
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Claims (4)
1.在翻译过程中直接实现蛋白质脂肪酸修饰的表达系统,其特征在于包括:
(1)脂肪酸类非天然氨基酸ε-N-庚酰基赖氨酸HepoK的盐酸盐或三氟乙酸盐,其分子结构式如下:
(2)编码对HepoK特异的正交氨酰tRNA合成酶的质粒;所述正交氨酰tRNA合成酶为突变合成酶1,其氨基酸序列为:
MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMMAPNLANYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFFQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL;
(3)表达宿主。
2.如权利要求1所述的表达系统,其特征在于编码突变合成酶1的核苷酸序列为:
ATGGATAAAAAGCCTTTGAACACTCTGATTTCTGCGACCGGTCTGTGGATGTCCCGCACCGGCACCATCCACAAAATCAAACACCATGAAGTTAGCCGTTCCAAAATCTACATTGAAATGGCTTGCGGCGATCACCTGGTTGTCAACAACTCCCGTTCTTCTCGTACCGCTCGCGCACTGCGCCACCACAAATATCGCAAAACCTGCAAACGTTGCCGTGTTAGCGATGAGGACCTGAACAAATTCCTGACCAAAGCTAACGAGGATCAGACCTCCGTAAAAGTGAAGGTAGTAAGCGCTCCGACCCGTACTAAAAAGGCTATGCCAAAAAGCGTGGCCCGTGCCCCGAAACCTCTGGAAAACACCGAGGCGGCTCAGGCTCAACCATCCGGTTCTAAATTTTCTCCGGCGATCCCAGTGTCCACCCAAGAATCTGTTTCCGTACCAGCAAGCGTGTCTACCAGCATTAGCAGCATTTCTACCGGTGCTACCGCTTCTGCGCTGGTAAAAGGTAACACTAACCCGATTACTAGCATGTCTGCACCGGTACAGGCAAGCGCCCCAGCTCTGACTAAATCCCAGACGGACCGTCTGGAGGTGCTGCTGAACCCAAAGGATGAAATCTCTCTGAACAGCGGCAAGCCTTTCCGTGAGCTGGAAAGCGAGCTGCTGTCTCGTCGTAAAAAGGATCTGCAACAGATCTACGCTGAGGAACGCGAGAACTATCTGGGTAAGCTGGAGCGCGAAATTACTCGCTTCTTCGTGGATCGCGGTTTCCTGGAGATCAAATCTCCGATTCTGATTCCGCTGGAATACATTGAACGTATGGGCATCGATAATGATACCGAACTGTCTAAACAGATCTTCCGTGTGGATAAAAACTTCTGTCTGCGTCCGATGATGGCGCCGAACCTTGCTAACTATGCTCGTAAACTGGACCGTGCCCTGCCGGACCCGATCAAAATTTTCGAGATCGGTCCTTGCTACCGTAAAGAGTCCGACGGTAAAGAGCACCTGGAAGAATTCACCATGCTGAACTTCTTCCAGATGGGTAGCGGTTGCACGCGTGAAAACCTGGAATCCATTATCACCGACTTCCTGAATCACCTGGGTATCGATTTCAAAATTGTTGGTGACAGCTGTATGGTGTATGGCGATACGCTGGATGTTATGCACGGCGATCTGGAGCTGTCTTCCGCAGTAGTGGGCCCAATCCCGCTGGATCGTGAGTGGGGTATCGACAAACCTTGGATCGGTGCGGGTTTTGGTCTGGAGCGTCTGCTGAAAGTAAAACACGACTTCAAGAACATCAAACGTGCTGCACGTTCCGAGTCCTATTACAATGGTATTTCTACTAACCTGTAA。
3.权利要求1或2所述表达系统在制备含HepoK的胰高血糖素样肽-1(GLP-1)中的应用。
4.一种脂肪酸修饰的药物蛋白的制备方法,其特征在于,所述药物蛋白为人胰高血糖素样肽-1(GLP-1),HepoK定点取代第26位赖氨酸;所述制备方法包括以下步骤:
(1)合成脂肪酸链非天然氨基酸ε-N-庚酰基赖氨酸HepoK的盐酸盐或者三氟乙酸盐,分子结构式如下:
(2)将蛋白基因特定位点DNA编码突变为TAG,获得药物蛋白表达载体;
(3)将药物蛋白表达载体与对HepoK特异的正交氨酰tRNA合成酶及正交tRNA表达载体共转,在大肠杆菌、酵母或哺乳动物细胞表达系统中制备带HepoK的药物蛋白;所述正交氨酰tRNA合成酶为突变合成酶1,其氨基酸序列为:
MDKKPLNTLISATGLWMSRTGTIHKIKHHEVSRSKIYIEMACGDHLVVNNSRSSRTARALRHHKYRKTCKRCRVSDEDLNKFLTKANEDQTSVKVKVVSAPTRTKKAMPKSVARAPKPLENTEAAQAQPSGSKFSPAIPVSTQESVSVPASVSTSISSISTGATASALVKGNTNPITSMSAPVQASAPALTKSQTDRLEVLLNPKDEISLNSGKPFRELESELLSRRKKDLQQIYAEERENYLGKLEREITRFFVDRGFLEIKSPILIPLEYIERMGIDNDTELSKQIFRVDKNFCLRPMMAPNLANYARKLDRALPDPIKIFEIGPCYRKESDGKEHLEEFTMLNFFQMGSGCTRENLESIITDFLNHLGIDFKIVGDSCMVYGDTLDVMHGDLELSSAVVGPIPLDREWGIDKPWIGAGFGLERLLKVKHDFKNIKRAARSESYYNGISTNL。
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Effective date of registration: 20220127 Address after: 310000 room 607, building 6, No. 600, Baiyang street, Qiantang District, Hangzhou City, Zhejiang Province Patentee after: Hangzhou Zhongke physicochemical Biomedical Technology Co.,Ltd. Address before: 310000 No. 600, 21st Street, Hangzhou Economic and Technological Development Zone, Zhejiang Province Patentee before: Technical Institute of Physics and Chemistry Chinese Academy of Sciences |