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  • FAQ: What are the strain properties (C2529)? 

    The properties of this strain that contribute to its usefulness as a protein expression strain are described below.

    T7 RNA Polymerase: T7 gene1 is encoded by the lambda DE3 prophage present within the chromosome. T7 RNA polymerase is expressed from the lacUV5 promoter, which is less sensitive to catabolite repression than the wt lac promoter. Thus DE3 strains may exhibit uninduced target protein expression. Although λ DE3 is normally dormant in the host chromosome, the induction of the SOS response can occur as the result of expressing proteins that damage the E. coli chromosome, either directly or indirectly. This may lead to cell lysis. T7 Express strains do not carry the DE3 prophage and better tolerate an SOS response.

    Protease Deficient ([lon] ompT): E. coli B strains are "naturally" deficient in the Lon protease which in K-12 strains serves to degrade misfolded proteins and to prevent some cell cycle-specific proteins from accumulating. The OmpT protease resides at the surface of wild type E. coli in both K-12 and B strains, presumably helping the cells to derive amino acids from their external environment. Cells deficient in both these proteases are much more amenable to the production of proteins from cloned genes.

    T1 Phage Resistant (fhuA2): T1, an extremely virulent phage requires the E. coli ferric hydroxamate uptake receptor for infectivity. Deletion of this gene confers resistance to this type of phage, but does not significantly affect the transformation or growth characteristics of the cell. 

    glmS6Ala: The glmS gene (glucosamine synthetase) is mutated and the expressed GlmS protein (67 kDa) contains six histidine to alanine substitutions (positions 62, 65, 432, 436, 466, 467). The mutated GlmS protein does not bind Ni-NTA resin in the presence of 20mM imidazole binding/ wash buffer, whereas wt GlmS protein binds Ni-NTA resin and is not eluted until the imidazole concentration is within 55–80 mM (1).

    arnA::CBD The arnA gene (lipid A modification) is fused with a B. circulans ORF encoding a chitin binding domain. The expressed fusion protein is 82 kDa whereas the wt ArnA protein is 74 kDa.

    can::CBD The can gene (carbonic anydrase) is fused with a B. circulans ORF encoding a chitin binding domain. The expressed fusion protein is 32 kDa whereas wt carbonic anhydrase is 25 kDa.

    slyD::CBD The slyD gene (FKBP-type prolyl isomerase) is fused with a B. circulans ORF encoding a chitin binding domain. The expressed fusion protein is 28 kDa. However, the apparent mass of the CBD fusion is approximately 35 kDa when analyzed by SDS-PAGE. The calculated mass of wt SlyD is 21 kDa, whereas the apparent mass is 28 kDa.

    The GlmS(6Ala) protein and the CBD-tagged proteins are functional in vivo according to assays conducted at New England Biolabs.