Technologies Available for License

2002-021: Auto-Induction in T7 Gene Expression Cells

Invention: 2002-021

Patent Status: U.S. Patent Number 7,704,722 was issued on April 27, 2010

For technical and licensing related questions, email tcp@bnl.gov.

Summary

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Electrophoresis of total cell proteins during growth of auto-inducing cultures at 37 ºC. In each set, equal culture densities were analyzed (A600: ~10 in A; ~10 in B; ~5, C; and ~2.5, D). The A600 of the culture at the time of sampling is given above each lane. (A) BL21(DE3)RIL/P21 was grown in 6 ml ZYP and 0.5% lactose in a 125-ml Erlenmeyer flask. (B) BL21(DE3)RIL/P21 was grown in 5 ml ZYP-5052 in a 125-ml flask (except that the glycerol concentration was 0.625% instead of 0.5%). (C) BL21(DE3)T7-10A was grown in 2.5 ml ZYP-20052 and 25 mM succinate in a 125-ml flask (the glycerol concentration was 2%). (D) BL21-AI/T7-10A was grown in 2 ml ZYM-5052 and 0.05% L-arabinose in an 18 X 150 mm culture tube.

Media in which T7 expression strains grow without induction and then transition automatically to a fully induced state without intervention have been developed. The media 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. 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. Large numbers of expression strains can 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 cultures all growing logarithmically at the same density for parallel testing in 96-well plates (or as many strains as desired for any parallel screening).

Description

The claims of this patent include methods for culturing expression host cells in media that are designed to permit the uninduced growth of expression strains followed by automatic induction of expression of target proteins and continued growth to saturation. The various media are available commercially or may be made from the details in the patent specification.

Journal Publication & Intellectual Property

• Protein production by auto-induction in high-density shaking cultures (.pdf)
• US 7,704,722 B2 (.pdf)

Press Releases

• Brookhaven Lab Biophysicist F. William Studier Wins R&D 100 Awared for A New Method that Simplifies Protein Production (.pdf)