Rapid preparation of bioluminescent tracers for relaxin family peptides using sortase-catalysed ligation
Rapid preparation of bioluminescent tracers for relaxin family peptides using sortase-catalysed ligation
复制标题
使用分选酶催化连接快速制备松弛素家族肽的生物发光示踪剂
DOI：
10.1007/s00726-017-2455-9
复制
发表时间：
2017
期刊：
影响因子：
3.5
通讯作者：
Guo Zhan-Yun
中科院分区：
文献类型：
--
作者：
Wang Jia-Hui;Shao Xiao-Xia;Hu Meng-Jun;Wei Dian;Nie Wei-Han;Liu Ya-Li;Xu Zeng-Guang;Guo Zhan-Yun
松弛素家族是一类通过激活G蛋白偶联受体RXFP 1 -4而发挥多种生物学功能的肽类激素。我们最近开发了生物发光示踪剂，通过与超灵敏的NanoLuc报告分子的化学结合进行受体结合测定。为了简化生物发光示踪剂的制备，在本研究中，我们建立了一个分选酶催化的连接方法使用嵌合R3/I5作为模型。在重组分选酶A催化后，通过NanoLuc的C-末端LPET序列和R3/I5的A-链N-末端Gly残基之间形成肽键，将在C-末端携带LPETG分选酶识别基序的NanoLuc报告基因有效连接到在A-链N-末端携带四个连续Gly残基的R3/I5肽。饱和结合试验表明，NanoLuc-ligated R3/I5保留了对RXFP 3和RXFP 4的高结合亲和力，计算的解离常数（Kd）分别为4.34 ± 0.33 nM（n= 3）和5.66 ± 0.54 nM（n= 3）。在竞争结合测定中使用NanoLuc-ligated R3/I5作为示踪剂，方便地定量各种配体对RXFP 3和RXFP 4的结合效力。本工作为松弛素家族肽和其他蛋白质/肽类激素的配体-受体相互作用研究提供了一种快速制备生物发光示踪剂的简单方法。
Relaxin family is a group of peptide hormones with a variety of biological functions by activating G protein-coupled receptors RXFP1–4. We recently developed bioluminescent tracers for their receptor-binding assays by chemical conjugation with the ultrasensitive NanoLuc reporter. To simplify preparation of the bioluminescent tracers, in the present study, we established a sortase-catalysed ligation approach using the chimeric R3/I5 as a model. Following catalysis by recombinant sortase A, a NanoLuc reporter carrying the LPETG sortase recognition motif at the C-terminus was efficiently ligated to an R3/I5 peptide carrying four successive Gly residues at the A-chain N-terminus, via the formation of a peptide bond between the C-terminal LPET sequence of NanoLuc and the A-chain N-terminal Gly residue of R3/I5. Saturation binding assays demonstrated that the NanoLuc-ligated R3/I5 retained high binding affinity to RXFP3 and RXFP4, with the calculated dissociation constants (Kd) of 4.34 ± 0.33 nM (n= 3) and 5.66 ± 0.54 nM (n= 3), respectively. Using the NanoLuc-ligated R3/I5 as a tracer in competition binding assays, binding potencies of various ligands towards RXFP3 and RXFP4 were conveniently quantified. This work provides a simple method for rapid preparation of bioluminescent tracers for relaxin family peptides and other protein/peptide hormones for ligand–receptor interaction studies.