Fernandes Cláudia S M, Dos Santos Raquel, Ottengy Stella, Viecinski Aline Canani, Béhar Ghislaine, Mouratou Barbara, Pecorari Frédéric, Roque A Cecília A
UCIBIO, REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade NOVA de Lisboa, 2829-516 Caparica, Portugal.
CRCNA, INSERM, CNRS, Université d'Angers, Université de Nantes, Nantes, France.
J Chromatogr A. 2016 Jul 29;1457:50-8. doi: 10.1016/j.chroma.2016.06.020. Epub 2016 Jun 7.
Currently most economical and technological bottlenecks in protein production are placed in the downstream processes. With the aim of increasing the efficiency and reducing the associated costs, various affinity ligands have been developed. Affitins are small, yet robust and easy to produce, proteins derived from the archaeal extremophilic "7kDa DNA-binding" protein family. By means of combinatorial protein engineering and ribosome display selection techniques, Affitins have shown to bind a diversity of targets. In this work, two previously developed Affitins (anti-lysozyme and anti-IgG) were immobilized onto magnetic particles to assess their potential for protein purification by magnetic fishing. The optimal lysozyme and human IgG binding conditions yielded 58mg lysozyme/g support and 165mgIgG/g support, respectively. The recovery of proteins was possible in high yield (≥95%) and with high purity, namely ≥95% and 81%, when recovering lysozyme from Escherichia coli supernatant and IgG from human plasma, respectively. Static binding studies indicated affinity constants of 5.0×10(4)M(-1) and 9.3×10(5)M(-1) for the anti-lysozyme and anti-IgG magnetic supports. This work demonstrated that Affitins, which can be virtually evolved for any protein of interest, can be coupled onto magnetic particles creating novel affinity adsorbents for purification by magnetic fishing.
目前，蛋白质生产中最主要的经济和技术瓶颈在于下游加工过程。为了提高效率并降低相关成本，人们开发了各种亲和配体。亲和体是一类体积小、稳定性高且易于生产的蛋白质，它们源自古细菌嗜极端微生物的“7kDa DNA结合”蛋白家族。通过组合蛋白质工程和核糖体展示筛选技术，亲和体已被证明能够结合多种靶标。在这项工作中，将两种先前开发的亲和体（抗溶菌酶亲和体和抗IgG亲和体）固定在磁性颗粒上，以评估它们通过磁捕法进行蛋白质纯化的潜力。溶菌酶和人IgG的最佳结合条件分别产生了58mg溶菌酶/克载体和165mg IgG/克载体的结合量。从大肠杆菌上清液中回收溶菌酶以及从人血浆中回收IgG时，蛋白质的回收率很高（≥95%）且纯度很高，分别为≥95%和81%。静态结合研究表明，抗溶菌酶和抗IgG磁性载体的亲和常数分别为5.0×10⁴M⁻¹和9.3×10⁵M⁻¹。这项工作表明，几乎可以针对任何感兴趣的蛋白质进行改造的亲和体，可以与磁性颗粒偶联，从而创建用于磁捕法纯化的新型亲和吸附剂。
