Stable expression and purification of a secreted human recombinant prethrombin-2 and its activation to thrombin.
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.
Russo
G
G
Dipartimento di Biochimica e Biotecnologie Mediche, Università di Napoli Federico II, Italy.
Gast
A
A
Schlaeger
E J
EJ
Angiolillo
A
A
Pietropaolo
C
C
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D004792:Enzyme Precursors / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D013058:Mass Spectrometry; D011516:Prothrombin / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D013917:Thrombin / Q000378:metabolism*; D014170:Transformation, Genetic
D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D011516:Prothrombin; C012007:prethrombins; D013917:Thrombin