Combination of ribosome display and next generation sequencing as a powerful method for identification of affibody binders against β-lactamase CTX-M15
Combination of ribosome display and next generation sequencing as a powerful method for identification of affibody binders against β-lactamase CTX-M15
复制标题
DOI：
10.1016/j.nbt.2019.01.004
复制
发表时间：
2019-05-25
影响因子：
5.4
通讯作者：
Stadthagen, Gustavo
中科院分区：
文献类型：
--
作者：
Lagoutte, Priscillia;Lugari, Adrien;Stadthagen, Gustavo
CTX-M15是感染人和动物的肠杆菌株中分布最广泛的超广谱β-内酰胺酶之一，是抗生素耐药性的主要决定因素，代表着紧迫的公共卫生威胁。在这里，我们描述了从展示在核糖体上的组合亲和体库中选择CTX-M15的结合子。经过三次选择性越来越高的核糖体展示迭代，通过下一代测序(NGS)确定了选定的变体。筛选出9个相对丰度较高的亲和体变异体，分别在第9~11位含有QRP和QLH型氨基酸基序，并对其稳定性、亲和力和特异性进行了研究。所有亲和抗体均被正确折叠，对CTX-M15的亲和力为0.04~2mM，并在细菌裂解物、培养上清液和整个细菌上成功识别CTX-M15。进一步证明，CTX-M15与亲和体分子的结合调节了酶的动力学参数。这项工作提供了一种利用核糖体展示与NGS偶联来快速产生诊断和研究应用中感兴趣的蛋白质配体的方法。
CTX-M15 is one of the most widespread, extended spectrum beta-lactamases, a major determinant of antibiotic resistance representing urgent public health threats, among enterobacterial strains infecting humans and animals. Here we describe the selection of binders to CTX-M15 from a combinatorial affibody library displayed on ribosomes. Upon three increasingly selective ribosome display iterations, selected variants were identified by next generation sequencing (NGS). Nine affibody variants with high relative abundance bearing QRP and QLH amino acid motifs at residues 9-11 were produced and characterized in terms of stability, affinity and specificity. All affibodies were correctly folded, with affinities ranging from 0.04 to 2 mu M towards CTX-M15, and successfully recognized CTX-M15 in bacterial lysates, culture supernatants and on whole bacteria. It was further demonstrated that the binding of affibody molecules to CTX-M15 modulated the enzyme's kinetic parameters. This work provides an approach using ribosome display coupled to NGS for the rapid generation of protein ligands of interest in diagnostic and research applications.