Purification and characterization of recombinant full-length and protease domain of murine MMP-9 expressed in Drosophila S2 cells.
Matrix metalloproteinase-9 (MMP-9) is a 92-kDa soluble pro-enzyme implicated in pathological events including cancer invasion. It is therefore an attractive target for therapeutic intervention studies in mouse models. Development of inhibitors requires sufficient amounts of correctly folded murine MMP-9. Constructs encoding zymogens of full-length murine MMP-9 and a version lacking the O-glycosylated linker region and hemopexin domains were therefore generated and expressed in stably transfected Drosophila S2 insect cells. After 7 days of induction the expression levels of the full-length and truncated versions were 5 mg/l and 2 mg/l, respectively. The products were >95% pure after gelatin Sepharose chromatography and possessed proteolytic activity when analyzed by gelatin zymography. Using the purified full-length murine MMP-9 we raised polyclonal antibodies by immunizations of rabbits. These antibodies specifically identified pro-MMP-9 in incisional skin wound extracts from mice when used for Western blotting. Immunohistochemical analysis of paraffin embedded skin wounds from mice showed that MMP-9 protein was localized at the leading-edge keratinocytes in front of the migrating epidermal layer. No immunoreactivity was observed when the antibody was probed against skin wound material from MMP-9 deficient mice. In conclusion, we have generated and purified two proteolytically active recombinant murine MMP-9 protein constructs, which are critical reagents for future cancer drug discovery studies.
Rasch
Morten G
MG
The Finsen Laboratory, Rigshospitalet Section 3735, Copenhagen Biocenter, Ole Maaløes Vej 5, Building 3.3, DK-2200 Copenhagen N, Denmark. mrasch@finsenlab.dk
Lund
Ida K
IK
Illemann
Martin
M
Høyer-Hansen
Gunilla
G
Gårdsvoll
Henrik
H
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002460:Cell Line; D002846:Chromatography, Affinity; D004330:Drosophila / Q000166:cytology / Q000235:genetics*; D015870:Gene Expression; D020780:Matrix Metalloproteinase 9 / Q000032:analysis / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification*; D051379:Mice; D051189:Murinae / Q000235:genetics*; D017434:Protein Structure, Tertiary; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D012867:Skin / Q000378:metabolism
D000906:Antibodies; D011994:Recombinant Proteins; D020780:Matrix Metalloproteinase 9