Biosynthesis and distribution of leucocyte elastase inhibitor. Production of recombinant inhibitor.
The horse leucocyte elastase inhibitor (HLEI), present in neutrophils, monocytes and bone marrow cells, is apparently a cytoplasmic protein which is not released from cells even in response to stimulation with lipopolysaccharide, phorbol ester, tumour necrosis factor alpha, interleukin-1 or elastin degradation products. Although no expression of the inhibitor was detected in neutrophils, both monocytes and bone marrow cells were efficient in its synthesis. Using a new expression vector pREST5d, recombinant inhibitor was produced in a large quantity in a soluble form, with a yield of 88 mg per 10 litres of E. coli culture. A two-step purification procedure, consisting of ion-exchange chromatography and gel filtration, yielded 36 mg of the recombinant inhibitor of a purity higher than 95%, as judged by SDS/PAGE. The recombinant protein had physicochemical and kinetic properties indistinguishable from those of the natural one, including irreversible elastase inhibition with an association rate constant kass > 10(7) M-1s-1. Both proteins were eliminated from rat circulation at the same ratio, and within the first 20 min 70% of the protein was removed. Such a short half-life in the circulation suggests that local delivery of HLEI directly to lungs in the form of aerosol could be a more efficient therapeutic approach than its intravenous injection.
Kasza
A
A
Institute of Molecular Biology, Jagiellonian University, Cracow, Poland.
Korpula-Mastalerz
R
R
Rose-John
S
S
Dubin
A
A
D000818:Animals; D004791:Enzyme Inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006736:Horses; D019272:Leukocyte Elastase / Q000037:antagonists & inhibitors*; D051381:Rats; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism
D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D019272:Leukocyte Elastase