基于组合策略的肽亲和色谱法用于蛋白质纯化。

Peptide affinity chromatography based on combinatorial strategies for protein purification.

作者信息

Camperi Silvia Andrea, Martínez-Ceron María Camila, Giudicessi Silvana Laura, Marani Mariela Mirta, Albericio Fernando, Cascone Osvaldo

机构信息

Institute of Nanobiotechnology UBA-CONICET, Cathedra of Industrial Microbiology and Biotechnology, School of Pharmacy and Biochemistry, University of Buenos Aires, Junín 956, C1113AAD, Buenos Aires, Argentina,

出版信息

Methods Mol Biol. 2014;1129:277-302. doi: 10.1007/978-1-62703-977-2_22.

DOI:10.1007/978-1-62703-977-2_22

PMID:24648083

Abstract

We describe a method to develop affinity chromatography matrices with short peptide ligands for protein purification. The method entitles the following: (a) synthesis of a combinatorial library on the hydromethylbenzoyl (HMBA)-ChemMatrix resin by the divide-couple-recombine (DCR) method using the Fmoc chemistry, (b) library screening with the protein of interest labeled with a fluorescent dye or biotin, (c) identification of peptides contained on positive beads by tandem matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS/MS), (d) solid-phase peptide ligand synthesis and immobilization in chromatographic supports, and (e) evaluation of protein adsorption on peptide affinity matrices from the equilibrium isotherms and breakthrough curves.

摘要

我们描述了一种开发带有短肽配体的亲和色谱基质用于蛋白质纯化的方法。该方法包括以下步骤：(a) 使用Fmoc化学方法，通过分割-偶联-重组（DCR）法在羟甲基苯甲酰（HMBA）-ChemMatrix树脂上合成组合文库；(b) 用荧光染料或生物素标记的目标蛋白对文库进行筛选；(c) 通过串联基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS/MS）鉴定阳性珠上所含的肽；(d) 固相肽配体的合成及固定于色谱支持物上；(e) 根据平衡等温线和穿透曲线评估蛋白质在肽亲和基质上的吸附情况。