TECHNOLOGY BRIEF

HIGH DENSITY GROWTH OF T7 EXPRESSION STRAINS WITH AUTO-INDUCTION OPTION

For further information, contact Christine Brakel

Description and Applications

Stable Growth and Maintenance of Expression Strains: By systematic optimization of media components, defined and complex media were developed that grow T7 expression strains to saturation at relatively high density with little or no induction of target protein. The pH of the cultures growing in these media remains in the range of 6-7 all the way to saturation, subcultures grow with little if any lag and freezer stocks appear to be stable indefinitely. These media are useful for preparing, maintaining and storing a wide range of T7 expression cultures, even those that express proteins quite toxic to the host. Many strains can be grown in parallel with no need to monitor growth of each and make freezer stocks from cultures still in log phase.

Auto-induction of Protein Production in the T7 Expression System in E. coli: A further systematic analysis was made to develop media in which T7 expression strains would grow initially without induction but then transition reliably to a fully induced state without intervention. The host was BL21(DE3) or B834(DE3) and the target genes were transcribed from a T7lac promoter. By adjusting the concentrations of mixtures of carbon sources, recipes were developed for media that allow growth of expression strains to relatively high densities with little or no production of target protein, followed by high level induction and growth to saturation at moderate pH. In these auto-inducing media, cultures saturate fully induced at optical densities typically in the range of 5-20 at 600 nm, and the yield of target protein per volume of culture is often 5-20 times that of a conventional induction. Thus, almost fermentor-type yields can be obtained in convenient shake flasks. A big advantage of these auto-inducing media is that they allow large numbers of expression strains to be analyzed in parallel for level of expression and solubility of the target protein, simply by inoculating and growing to saturation. Cultures do not have to be monitored, inducer does not have to be added at the proper time and there is no necessity to obtain 96 strains all growing logarithmically at the same density for parallel testing in 96-well plates (or as many strains as desired for any parallel screening).

Selenomethionine labeling of proteins for crystallography: Proteins labeled with selenomethionine are useful for determining structures of proteins by X-ray crystallography, but obtaining good yields of the labeled protein has often been difficult. Supplementing defined, auto-inducing medium was found to significantly increase the yield of selenomethionine-labeled protein. It is likely that the supplement activates an enzyme required to regenerate selenomethionine that may have been metabolized to selenohomocysteine. Yields of selenomethionine-labeled protein in the supplemented auto-inducing medium are usually almost as great as obtained with normal protein.

Inventors: F. William Studier

Patent Status: U.S. Patent Application Serial No.10/675936 pending.

License Status: Available, non-exclusively, on a negotiable basis for research and commercial use.