Synthesis of heterobifunctional protein fusions using copper-free click chemistry and the aldehyde tag.
Synthesis of heterobifunctional protein fusions using copper-free click chemistry and the aldehyde tag.
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DOI：
10.1002/anie.201108130
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发表时间：
2012-04-23
影响因子：
16.6
通讯作者：
Rabuka, David
中科院分区：
文献类型：
--
作者：
Hudak, Jason E.;Barfield, Robyn M.;de Hart, Gregory W.;Grob, Patricia;Nogales, Eva;Bertozzi, Carolyn R.;Rabuka, David
异双功能蛋白融合体作为下一代生物药物正在引起人们的兴趣。[1-5]将具有不同功能的蛋白质组合可以使单一化学实体的多药治疗成为可能，[6，7]将靶向元件添加到其他非特异性治疗剂中，[8，9]或改善快速清除分子的药代动力学特征。[10事实上，异型双功能蛋白质，如免疫球蛋白G（IgG）Fc结构域融合物，是当今市场上最畅销的生物治疗剂之一。[12]这些生物分子主要是作为基因融合产生的。编码单个蛋白质组分的DNA序列串联融合以指导单个多肽的表达，所述单个多肽包含分别在其N和C末端连接在一起的两种蛋白质。然而，这种有限的拓扑结构对于每种蛋白质组合都不是理想的，因为一些多肽需要未修饰的末端以获得最佳生物活性[13]，或者由于折叠和加工问题而可能遭受表达困难。[3，14，15]产生蛋白质-蛋白质融合的另一种方法是通过化学缀合。C-末端硫酯与β-氨基硫醇的天然化学连接是产生蛋白质-蛋白质融合物的有效方法[16-18]，但必须在其末端连接至少一个偶联配偶体。原则上，可以通过在两种蛋白质的特定氨基酸侧链处引入生物正交官能团来实现更大的拓扑多样性。[19，20]作为最近的例子，Hutchins埃塔尔。表达带有非天然酮氨基酸的Fab片段，马来酰亚胺官能化接头通过肟形成与该非天然酮氨基酸缀合。[21]这反过来又使得能够进一步缀合到被工程化到蛋白质毒素中的单个半胱氨酸残基。在这项工作中隐含的是需要一个蛋白质-蛋白质偶联反应与固有的快速动力学，其中硫醇马来酰亚胺加成是一个典范的例子。在这个方向上，Bundy和Swartz实现了无细胞蛋白质合成，将叠氮和炔氨基酸安装到绿色荧光蛋白中，用于Cu催化的二聚化。[22]然而，这种方法遭受低蛋白表达以及铜诱导的蛋白质损伤。环辛炔和叠氮化物的应变促进的1，3-偶极环加成，也称为无Cu叠氮化物-炔环加成，是非常适合于蛋白质-蛋白质缀合的生物正交反应。[23-26]环辛炔试剂可以被调节用于快速动力学，并且反应在宽范围的条件下选择性地进行。[27-31]然而，利用这些特性进行异型双功能蛋白质缀合物合成首先需要一种用于位点特异性引入必要的反应性配偶体的实用途径。遗传编码的醛标签提供了一种简单的位点特异性蛋白质功能化的方法。[32-34]该标签由一个简洁的五个残基序列（CxPxR）组成，该序列被甲酰甘氨酸生成酶（FGE）识别。FGE在蛋白质表达过程中将基因编码的半胱氨酸残基氧化为带有酰化酶的残基甲酰甘氨酸（fGly）。大肠杆菌或哺乳动物细胞（方案1A）。[35]然后可以通过形成腙或肟来修饰醛（方案1B）。[36]因此，醛标签作为一种手段，用于通过小分子量的寡核苷酸将叠氮化物或环辛炔位点特异性引入重组蛋白。
Heterobifunctional protein fusions are gaining interest as next-generation biopharmaceuticals.[1–5] Combining proteins with disparate functions can enable multidrug therapy with a single chemical entity,[6, 7] add a targeting element to an otherwise nonspecific therapeutic,[8, 9] or improve the pharmacokinetic profile of a rapidly cleared molecule.[10, 11] Indeed, heterobifunctional proteins, such as immunoglobulinG (IgG) Fc domain fusions, are among the top-selling biotherapeutics on the market today.[12] These biomolecules are primarily generated as genetic fusions. The DNA sequences that encode the individual protein components are fused in tandem to direct the expression of a single polypeptide that comprises the two proteins joined together at their N and C termini, respectively. However, this limited topology is not ideal for every protein combination, as some polypeptides require unmodified termini for optimal bioactivity [13] or can suffer from expression difficulties as a result of folding and processing issues.[3, 14, 15] An alternative approach to generating protein–protein fusions is through chemical conjugation. Native chemical ligation of C-terminal thioesters with β-amino thiols is a powerful method for generating protein–protein fusions,[16–18] but at least one coupling partner must be linked at its terminus. In principle, greater topological diversity can be achieved by introducing bioorthogonal functional groups at specific amino acid side chains of the two proteins.[19, 20] As a recent example, Hutchins etal. expressed a Fab fragment bearing an unnatural keto amino acid to which a maleimide-funtionalized linker was conjugated by oxime formation.[21] This, in turn, enabled further conjugation to a single cysteine residue that was engineered into a protein toxin. Implicit in this work is the need for a protein–protein coupling reaction with intrinsically fast kinetics, of which thiol to maleimide addition is a paragon example. In this direction, Bundy and Swartz have implemented cell-free protein synthesis to install azide and alkyne amino acids into green fluorescent protein for Cu-catalyzed dimerization.[22] This approach, however, suffered from low protein expression as well as Cu-induced protein damage. The strain-promoted 1, 3-dipolar cycloaddition of cyclooctynes and azides, also termed the Cu-free azide–alkyne cycloaddition, is a bioorthogonal reaction that is well suited for protein–protein conjugation.[23–26] The cyclooctyne reagent can be tuned for fast kinetics and the reaction proceeds selectively under a wide range of conditions.[27–31] However, harnessing these qualities for heterobifunctional protein conjugate synthesis first requires a practical route for the site-specific introduction of the necessary reactive partners. The genetically encoded aldehyde tag offers a simple means of site-specific protein functionalization.[32–34] The tag consists of a succinct five-residue sequence (CxPxR) that is recognized by the formylglycine-generating enzyme (FGE). FGE oxidizes the genetically-encoded cysteine residue to the aldehyde-bearing residue formylglycine (fGly) during protein expression in either E. coli or mammalian cells (Scheme 1 A).[35] The aldehyde can then be modified by hydrazone or oxime formation (Scheme1B).[36] Thus, the aldehyde tag serves as a means for site-specific introduction of azides or cyclooctynes onto recombinant proteins through small-mol-
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影响因子：
46.2
作者：
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通讯作者：
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影响因子：
100.3
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影响因子：
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作者：
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作者：
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