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Title
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Stable expression and purification of a secreted human recombinant prethrombin-2 and its activation to thrombin.
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Authors
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G.Russo,
A.Gast,
E.J.Schlaeger,
A.Angiolillo,
C.Pietropaolo.
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Ref.
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Protein Expr Purif, 1997,
10,
214-225.
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PubMed id
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Abstract
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A human prothrombin cDNA has been engineered to obtain a cDNA coding for a
secreted form of human prethrombin-2. The secreted prethrombin-2 has been
produced in a mammalian expression system using DXB11 cells, a mutant strain of
CHO cells in which the dihydrofolate reductase gene has been deleted, and an
expression vector carrying the dihydrofolate reductase cDNA.
Methotrexate-induced gene amplification favored an efficient production of the
recombinant protein which accumulated in the culture medium of the DXB11 cells.
Growth in suspension of the stable transformants in an airlift fermenter
resulted in the production of 25 mg/L recombinant prethrombin-2. The recombinant
protein was purified using single-step affinity chromatography on a
recombinant-hirudin column and activated by agarose gel-immobilized ecarin. All
purified recombinant prethrombin-2 was activated and the generated recombinant
thrombin showed catalytic properties identical to those of plasma-derived
alpha-thrombin. This expression system can be used to prepare mutants of
prethrombin-2 for structure-function studies investigating thrombin interactions
with substrate proteins, inhibitors, and cell membranes.
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