Isolation and purification of protein complexes of MT1-MMP-hTAP fusion gene using tandem affinity purification system in chick fibroblast cells and evaluation of the expression effect / 第三军医大学学报

ABSTRACT

Objective To construct the fusion gene of membrane type matrix metalloproteinase1 humanized tandem affinity purification (MT1 MMP hTAP) and to evaluate the expression of its protein complexes isolated and purified using tandem affinity purification (TAP) system. Methods The MT1 MMP hTAP protein complexes were isolated and purified using the IgG agarose beads and calmodulin sepharose beads affinity chromatography according to the molecular and biological features of TAP system. The expression levels of its protein complexes were determined by PAP immunostaining and Western blotting. Results PAP antibody immunostaining showed that MT1 MMP hTAP protein complexes (dark blue) were expressed in chick fibroblast cells. Western blot analysis of its protein complexes using MT1 MMP specific antibody showed a prominent band (68?10 3) of mobility consistent with fully processed MT1 MMP hTAP after being cleaved by the TEV proteinase. Conclusion The construction of TAP system in chick fibroblast cells and expression of MT1 MMP hTAP protein complexes will be helpful for the studies of the MT1 MMP hTAP interaction proteins in the human tumor cells.