title: Automated expression and solubility screening of His-tagged proteins in 96-well format.
abstract: 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.
authors.affiliation: Architecture et Fonction des Macromolécules Biologiques, UMR 6098, CNRS/Universités d'Aix-Marseille I et II, Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France.
authors.forename: Renaud
authors.initials: R
authors.lastname: Vincentelli
authors.forename: Stéphane
authors.initials: S
authors.lastname: Canaan
authors.forename: Julien
authors.initials: J
authors.lastname: Offant
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
authors.forename: Christophe
authors.initials: C
authors.lastname: Bignon
mesh_terms: D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry; D012371:Robotics; D012815:Signal Processing, Computer-Assisted; D012995:Solubility
chemical_list: D011994:Recombinant Proteins; D006639:Histidine