| 摘要 |
Apolipoprotein A1 is prepared in a T7 RNA polymerase/promoter bacterial expression system that optimizes yield and facilitates subsequent purification of the protein. Apolipoprotein A1 cDNA is cloned into an Escherichia coli plasmid containing a bacteriophage T7 RNA polymerase promoter such as a pT or pET plasmid; the recombinant plasmid is transferred to an E. coli strain containing inducible bacteriophage T7 RNA polymerase, e.g., a strain having the enzyme under lac control; T7 RNA polymerase is induced in the strain; and apolipoprotein A1 is isolated from the culture. Preferred embodiments tag the apolipoprotein with a sequence such as a stretch of histidines that facilitates purification subsequent to expression. An especially preferred embodiment employs a pET11d-6His plasmid and E. coli strain BL21(DE)pLysS that produces at least 1 mg protein per liter of culture, preferably up to 2 mg per liter, and the protein can be purified to homogeneity by one-step affinity chromatography on a nickel chelating resin. |
