Capturing protein-protein complexes at equilibrium:: The holdup comparative chromatographic retention assay

被引：19

作者：

Charbonnier, Sebastian ^{[1
]}

Zanier, Katia ^{[1
]}

Masson, Murielle ^{[1
]}

Trave, Gilles ^{[1
]}

机构：

[1] Ecole Super Biotechnol Strasbourg, UMR 7175, CNRS, Equipe Oncoprot,LCE, F-67412 Illkirch Graffenstaden, France

来源：

PROTEIN EXPRESSION AND PURIFICATION | 2006年 / 50卷 / 01期

基金：

澳大利亚研究理事会;

关键词：

rapid; easy; assay; protein-protein interaction; affinity chromatography; equilibrium constant; elusive complexes; fusion protein; GST; MBP;

D O I：

10.1016/j.pep.2006.06.010

中图分类号：

Q5 [生物化学];

学科分类号：

071010 [生物化学与分子生物学]; 081704 [应用化学];

摘要：

The popular pulldown chromatographic assay detects complexes mediated by fusion proteins retained on affinity resin. The main limitation of this method is that it does not analyze complexes at equilibrium but after several washing steps. Consequently, fast-dissociating complexes may remain undetected. Here, we present the holdup assay, based on the principle of comparative chromatographic retention which eliminates the use of washing steps. The assay evaluates fractions of free and bound species at equilibrium. We used human papillomavirus oncoprotein E6, an E6-binding peptide and an E6-binding PDZ domain, to test several protocols utilizing pure proteins or expression extracts. The holdup assay is faster and more informative than the pulldown assay. It detects fast-dissociating complexes and it is also suited for evaluating equilibrium constants. It is potentially adaptable for automated determination of affinity constants and high-throughput analysis of interactions between proteins and other proteins, peptides, nucleic acids, or small regulatory molecules. (c) 2006 Elsevier Inc. All rights reserved.

引用

页码：89 / 101

页数：13

共 29 条

[1]

IDENTIFICATION OF THE HPV-16 E6 PROTEIN FROM TRANSFORMED MOUSE CELLS AND HUMAN CERVICAL-CARCINOMA CELL-LINES [J].