Expression and secretion of active mouse TIMP-1 using a baculovirus expression vector.
Mouse TIMP-1, one of the tissue inhibitors of metalloproteinases important in regulating turnover of extracellular matrix in both normal and pathological tissues, was previously expressed in E. coli in an inactive, nonglycosylated state that required refolding to become functional. Due to the difficulty of renaturation, an alternative to the prokaryotic expression system was sought. Since we are interested in studying the pharmacodynamics and pharmacokinetics of TIMP locally administered by controlled delivery to mice with experimentally induced arthritis, we also needed an efficient way of producing active TIMP in large quantities. Using the pBlueBacII transfer vector, we generated a recombinant baculovirus that in Sf9 cells could express glycosylated mouse TIMP-1 to about 3 mg of active protein/liter conditioned medium.
Cocuzzi
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Department of Biological Sciences, Rutgers University, Piscataway, New Jersey 08855.
Walther
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Denhardt
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D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D005822:Genetic Vectors; D006023:Glycoproteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D009036:Moths; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D019714:Tissue Inhibitor of Metalloproteinases
D006023:Glycoproteins; D011994:Recombinant Proteins; D019714:Tissue Inhibitor of Metalloproteinases