High level production of secreted proteins: example of the human tissue inhibitor of metalloproteinases 1.
The major difficulty for high-throughput screening of therapeutic protein candidates in experimental animal models of pathologies or for structural studies is their fast and efficient production. The tissue inhibitors of metalloproteinases (TIMPs) considered to play a role in many physiological and pathological processes, such as arthritis or cancer, by inhibiting matrix metalloproteinases or acting as signalling molecules, have always been produced with huge difficulties. We hereby propose a new method to overproduce human recombinant TIMP-1 by transient expression in HEK293E cells, followed by a one-step chromatography purification, yielding in only 2 weeks, dozens of milligrams of pure, stable, glycosylated and active protein for in vitro and in vivo studies. This easy to set up, rapid, and efficient method could be applied for any naturally secreted mammalian protein.
Crombez
L
L
Groupe de Recherche et d'Etude du Processus Inflammatoire, MENRT EA 2938, DBPC/Enzymologie, Centre Hospitalier Universitaire de Grenoble, BP 217, 38043 Grenoble Cedex 9, France. laurence_crombez@yahoo.fr
Marques
B
B
Lenormand
J L
JL
Mouz
N
N
Polack
B
B
Trocme
C
C
Toussaint
B
B
D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods*; D024861:Genetic Enhancement / Q000379:methods; D006801:Humans; D007668:Kidney / Q000378:metabolism*; D015202:Protein Engineering / Q000379:methods*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis* / Q000302:isolation & purification*; D014162:Transfection / Q000379:methods*
D011994:Recombinant Proteins; D019715:Tissue Inhibitor of Metalloproteinase-1