Zhang Yi, Auger Shelby, Schaefer Jonas V, Plückthun Andreas, Distefano Mark D
Department of Chemistry, University of Minnesota, Minneapolis, MN, USA.
Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Methods Mol Biol. 2019;2033:207-219. doi: 10.1007/978-1-4939-9654-4_14.
Affinity agents coupled to a functional moiety play an ever-increasing role in modern medicine, ranging from radiolabeled selective binders in diagnosis to antibody-drug conjugates in targeted therapies. In biomedical research, protein coupling to fluorophores, surfaces and nanoparticles has become an integral part of many procedures. In addition to antibodies, small scaffold proteins with similar target binding properties are being widely explored as alternative targeting moieties. To label these binders of interest with different functional moieties, conventional chemical coupling methods can be employed, but often result in heterogeneously modified protein products. In contrast, enzymatic labeling methods are highly site-specific and efficient. Protein farnesyltransferase (PFTase) catalyzes the transfer of an isoprenoid moiety from farnesyl diphosphate (FPP) to a cysteine residue in a C-terminal CaaX motif at the C-terminus of a protein substrate. The addition of only four amino acid residues minimizes the influence on the native protein structure. In addition, a variety of isoprenoid analogs containing different bioorthogonal functional groups, including azides, alkynes, and aldehydes, have been developed to enable conjugation to various cargos after being incorporated onto the target protein by PFTase. In this protocol, we present a detailed procedure for labeling Designed Ankyrin Repeat Proteins (DARPins) engineered with a C-terminal CVIA sequence using an azide-containing FPP analog by yeast PFTase (yPFTase). In addition, procedures to subsequently conjugate the labeled DARPins to a TAMRA fluorophore using strained-promoted alkyne-azide cycloaddition (SPAAC) reactions as well as the sample preparation to evaluate the target binding ability of the conjugates by flow cytometry are described.
与功能部分偶联的亲和剂在现代医学中发挥着越来越重要的作用，从诊断中的放射性标记选择性结合剂到靶向治疗中的抗体 - 药物偶联物。在生物医学研究中，蛋白质与荧光团、表面和纳米颗粒的偶联已成为许多程序不可或缺的一部分。除了抗体之外，具有相似靶标结合特性的小型支架蛋白作为替代靶向部分也在被广泛探索。为了用不同的功能部分标记这些感兴趣的结合剂，可以采用传统的化学偶联方法，但往往会产生修饰不均一的蛋白质产物。相比之下，酶促标记方法具有高度的位点特异性和高效性。蛋白质法尼基转移酶（PFTase）催化类异戊二烯部分从法尼基二磷酸（FPP）转移到蛋白质底物C末端CaaX基序中的半胱氨酸残基上。仅添加四个氨基酸残基可将对天然蛋白质结构的影响降至最低。此外，已经开发出多种含有不同生物正交功能基团（包括叠氮化物、炔烃和醛）的类异戊二烯类似物，以便在通过PFTase掺入靶蛋白后能够与各种货物偶联。在本方案中，我们展示了使用酵母PFTase（yPFTase）通过含叠氮化物的FPP类似物标记经工程改造带有C末端CVIA序列的设计锚蛋白重复蛋白（DARPins）的详细步骤。此外，还描述了随后使用应变促进的炔烃 - 叠氮环加成（SPAAC）反应将标记的DARPins与TAMRA荧光团偶联的步骤以及通过流式细胞术评估偶联物靶标结合能力的样品制备方法。
