Medium-scale purification for expression-clone screening and protein characterization. A mixture of vector constructs for the expression of proteins of 12, 15, and 28 kDa, and a 48/50 kDa heterodimer was transformed into E. coli, plated on selective medium, and 96 colonies were picked at random for inoculating 5 ml cultures. Expression of 6xHis-tagged proteins was induced with IPTG overnight. Cells were pelleted and lysed and 6xHis-tagged proteins purified. 5 µl of each elution fraction was loaded for SDSPAGE analysis; proteins were visualized by Coomassie staining.
Vector constructs for the expression of His-tagged proteins were transformed into E. coli, plated on selective medium, and colonies were picked for inoculating 25 ml cultures. Expression of 6xHis-tagged proteins was induced with IPTG for 2–4 hours. Cells were pelleted in 24-well blocks and processed on the BioRobot 3000 using 200 µl Ni-NTA Superflow resin per well. 5 µl (0.9%) of the first elution fraction was loaded for SDS-PAGE and proteins were visualized by Coomassie staining. G: Green Fluorescent Protein (29 kDa); T: T7 RNA Polymerase (100 kDa); S: E. coli GroES (12 kDa). Some endogenous GroEL is copurified; C: E. coli chloramphenicol acetyltransferase (28 kDa); L: E. coli GroEL (60 kDa); α: human tumor necrosis factor α(18 kDa); E: E. coli GroES purified as a complex with co-overexpressed nontagged GroEL (12 and 60 kDa); 10: Saccharomyces cerevisiae Cpn-10 (10 kDa). M: markers.