Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins

被引：6

作者：

Kery, V ^{[1
]}

Savage, JR ^{[1
]}

Widjaja, K ^{[1
]}

Blake, BK ^{[1
]}

Conklin, DR ^{[1
]}

Ho, YSJ ^{[1
]}

Long, XH ^{[1
]}

von Rechenberg, M ^{[1
]}

Zarembinski, TI ^{[1
]}

Boniface, JJ ^{[1
]}

机构：

[1] Myriad Prote Inc, Salt Lake City, UT 84116 USA

来源：

ANALYTICAL BIOCHEMISTRY | 2003年 / 317卷 / 02期

关键词：

D O I：

10.1016/S0003-2697(03)00116-7

中图分类号：

Q5 [生物化学];

学科分类号：

071010 ; 081704 ;

摘要：

High-throughput purification of affinity-tagged fusion proteins is currently one of the fastest developing areas of molecular proteomics. A prerequisite for success in protein purification is sufficient soluble protein expression of the target protein in a heterologous host. Hence, a fast and quantitative evaluation of the soluble-protein levels in an expression system is one of the key steps in the entire process. Here we describe a high-throughput expression screen for affinity-tagged fusion proteins based on an enzyme linked immunofiltration assay (ELIFA). An aliquot of a crude Escherichia coli extract containing the analyte, an affinity-tagged protein, is adsorbed onto the membrane. Subsequent binding of specific antibodies followed by binding of a secondary antibody horseradish peroxidase (HRP) complex then allows quantitative evaluation of the analyte using tetramethylbenzidine as the substrate for HRP. The method is accurate and quantitative, as shown by comparison with results from western blotting and an enzymatic glutathione S-transferase (GST) assay. Furthermore, it is a far more rapid assay and less cumbersome than western blotting, lending itself more readily to high-throughput analysis. It can be used at the expression level (cell lysates) or during the subsequent purification steps to monitor yield of specific protein. (C) 2003 Elsevier Science (USA). All rights reserved.

引用

页码：255 / 258

页数：4

共 3 条

[1] Proteome-scale purification of human proteins from bacteria [J].