Autoinduction of Protein Expression

Abstract

This unit contains protocols for the use of lactose-derived autoinduction in Escherichia coli. The protocols allow for reproducible expression trials to be undertaken with minimal user intervention. A basic protocol covers production of unlabeled proteins for functional studies. Alternate protocols for selenomethionine labeling for X-ray structural studies, and multi-well plate growth for screening and optimization are also included.

Active Site and Laminarin Binding in Glycoside Hydrolase Family 55

“…Sequence-verified plasmids were transformed into either E. coli B834 (selenomethionine labeling) or E. coli BL21 (DE3), respectively, for heterologous expression (31,34). A single colony from an overnight transformation was inoculated into 1 ml of chemically defined noninducing medium containing 50 mg/liter kanamycin and 34 mg/liter chloramphenicol for 8 h at 37°C (31).…”

Prion Protein Prolines 102 and 105 and the Surrounding Lysine Cluster Impede Amyloid Formation

“…In brief, pET41 vector containing hamster (accession number K02234), mouse (accession number M13685), or mutant PrP c sequences was transformed into Rosetta (BL21DE3) Escherichia coli cells. Protein was expressed by autoinduction (32,33). Inclusion bodies were collected and solubilized in 8 M guanidine HCl.…”

Mutational Analysis of Quinolone Resistance Protein QnrB1

“…Wild-type and mutant QnrB1-His 6 proteins were purified as previously described (6). In brief, BL21(DE3) derivatives were grown in autoinduction medium (10), harvested by centrifugation, and treated with lysozyme and sonication. After centrifugation, the supernatant was applied to a nickel-nitrilotriacetic acid (Ni-NTA) column (HiTrap Chelating HP; GE Healthcare).…”