High yield production of human apolipoprotein A1 in E. coli.

Details High yield production of human apolipoprotein A1 in E. coli. High yield production of human apolipoprotein A1 in E. coli.

1994-03-21

1997-07-01

Walsh, Stephen

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Recombinant dna technique included in method of making a...

435 696, 43525233, 4353201, 935 39, 530413, 530415, C12N 1570, C07K 122

Patent

active

056437579

ABSTRACT:
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.

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