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Protocol Development for the Production of Purified Matrix Metalloproteinase 1
Author(s) -
DeMarco Angela Mary,
Grove Laurie
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.04406
Subject(s) - mmp1 , biochemistry , matrix metalloproteinase , metalloproteinase , chemistry , histidine , zinc , plasmid , amino acid , biology , microbiology and biotechnology , gene , gene expression , organic chemistry
Matrix metalloproteinase 1 (MMP1) is an enzyme involved in the degradation of the extracellular matrix of cells. In human MMPs, an active site containing three histidine amino acids uses zinc to catalyze peptide bond cleavage. Higher expression of this protein is associated with cancer and arthritis, so there is interest in using MMP1 to target drug delivery for these patients. Because there are twenty‐six MMPs, inhibiting the activity of one may influence the others causing unforeseen downstream affects. This is largely due to poor selectivity of the inhibitors, so more specificity is required to ensure only MMP1 is targeted. The simple multicellular organism Volvox uses proteinases that are more active with copper than zinc. Zinc and copper have similar acid/base properties, so they should function in a similar way but don’t within these two systems. Volvox has two histidines and one glutamine in its active site. Exploring the functionality of the two proteinases with different metals may reveal the importance of the three histidines in its preference to zinc. Before experimentation can occur on MMP1, a method to produce large amounts of purified protein needs to be established. Plasmids containing the gene for human full‐length proenzyme MMP1 with His tags was purchased and the identity was confirmed using restriction digest. These plasmids were then used to transform E. coli which was cultured to allow for protein expression. Sonication was used to lyse the cells. The lysate was then purified using Ni 2+ magnetic beads. SDS‐PAGE and kinetic assays were done on the MMP1 to ensure that it was an active enzyme sample. It was found that the E. coli was properly producing the protein, but further work is being done to ensure it is fully active. When this production method is perfected, the MMP1 collected here will be used in metal replacement assays and work with Crispr to generate multiple active site single point mutations. Support or Funding Information Wentworth Institute of Technology