Journal
ANALYTICAL BIOCHEMISTRY
Volume 346, Issue 1, Pages 77-84Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ab.2005.07.039
Keywords
expression screening; solubility screening; structural genomics; robotics; dot-blot; protein production; high-throughput
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A growing need for sensitive and high-throughput methods for screening the expression and solubility of recombinant proteins exists in structural genomics. Originally, the emergency solution was to use immediately available techniques such as manual lysis of expression cells followed by analysis of protein expression by gel electrophoresis. However, these handmade methods quickly proved to be unfit for the high-throughput demand of postgenomics, and it is now generally accepted that the long-term solution to this problem will be based on automation, on industrial standard-formatted experiments, and on downsizing samples and consumables. In agreement with this consensus, we have set up a fully automated method based on a dot-blot technology and using 96-well format consumables for assessing by immunodetection the amount of total and soluble recombinant histidine (His)-tagged proteins expressed in Escherichia coli. The method starts with the harvest of expression cells and ends with the display of solubility/expression results in milligrams of recombinant protein per liter of culture using a three-color code to assist analysis. The program autonomously processes 160 independent cultures at a time. (C) 2005 Elsevier Inc. All rights reserved.
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