Expression screen by enzyme-linked immunofiltration assay designed for high-throughput purification of affinity-tagged proteins.
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.
Kery
Vladimir
V
Myriad Proteomics, Inc., Salt Lake City, UT 84116, USA. vkery@myriad-proteomics.com
Savage
Justin R
JR
Widjaja
Kartika
K
Blake
B Kelly
BK
Conklin
David R
DR
Ho
Yew-Seng J
YS
Long
Xinghua
X
von Rechenberg
Moritz
M
Zarembinski
Thomas I
TI
Boniface
J Jay
JJ
D015153:Blotting, Western; D002846:Chromatography, Affinity / Q000379:methods*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015203:Reproducibility of Results
D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase