Production and purification of recombinant C-terminal truncated pro-Matrix Metalloprotease-9

Abstract

MMP-9 consists of N-terminal signal peptide, a pro-domain, catalytic domain, a long flexible hinge region and C- terminal hemopexin domain. In the present study a truncated version of proMMP-9 which lacking hinge region and C-terminal hemopexin domain was produced. To do so Sf9 insect cells and the BaculoDirect Baculovirus expression system were used. Using site directed mutagenesis, single mutation in the beginning of hinge region was introduced that resulted in a stop codon in the MMP-9 DNA. A recombinant Baculovirus was generated containing the Mutated MMP-9 DNA. By transfecting Sf9 cells with the recombinant Baculovirus genome a truncated version of MMP-9(proMMP-9ΔH-HPX) was produced. The proMMP-9ΔH-HPX was purified by Gelatin Sepharose chromatography and Size exclusion chromatography. Gelatin Zymography revealed the activity of proMMP-9ΔH-HPX. Western blot and Mass spectrometry confirmed the identity of proMMP-9ΔH-HPX. The purity of proMMP-9ΔH-HPX was established by SDS-PAGE and Coomassie staining