Expression, purification, refolding and in vitro recovery of active full length recombinant human gelatinase MMP-9 in Escherichia coli

July 28, 2017

Title

Expression, purification, refolding and in vitro recovery of active full length recombinant human gelatinase MMP-9 in Escherichia coli

Author

Sara Mohseni, Tahereh Tohidi Moghadam, Bahareh Dabirmanesh, Khosro Khajeh

Year

2016

Journal

Protein Expression and Purification

Abstract

Human gelatinase (MMP-9) is a member of matrix metalloproteinases family (MMPs), which has been associated with malignant tumor progression and metastasis by matrix degradation. Herein, active full length recombinant human MMP-9 (amino acid residues 107-707) has been expressed in the form of inclusion bodies in Escherichia coli BL21, using pET21a vector. Solubilization of inclusion bodies was carried out in Tris-HCl buffer with 6 M urea, and refolding was performed using dilution and urea gradient methods. Tris-HCl buffer with 5 mM CaCl2 and 1 µM ZnCl2 at pH 7.8 was used as a refolding buffer. Analysis of the structure by fluorescence and far-UV circular dichroism showed a well-formed structure by urea gradient method. Kinetic parameters in refolding conditions of rhMMP-9 were also analyzed, depicting increase in the enzyme’s activity without any aggregation.

Instrument

J-715

Keywords

Circular dichroism, Secondary structure, Protein folding, Protein denaturation, Biochemistry, Pharmaceutical