CoESPRIT: A Library-Based Construct Screening Method for Identification and Expression of Soluble Protein Complexes
Figure 2
Process of screening for soluble protein complexes from truncation libraries.
9,216 colonies were picked for each library and gridded onto nitrocellulose membranes over agar to generate colony arrays in which protein expression was induced. Expression levels and putative solubility were assessed by intensity of PB2 N-terminal hexahistidine tag (green) and C-terminal biotinylated biotin acceptor peptide (red) signals respectively on the colony blot. The 96 highest ranked clones were expressed in 4 ml liquid expression cultures, purified by Ni2+NTA affinity chromatography and eluted fractions analysed by western blot to identify bait FLAG tag (blue) and PB2 biotinylation signals (red). From these data, a panel of clones was selected for 30 ml scale expression tests, then further prioritised for larger scale production.