Efficient protease based purification of recombinant matrix metalloprotease-1 in E. coli

• Published in: Protein expression and purification Vol. 148; pp. 59 - 67
• Main Authors: Kumar, Lokender, Colomb, Warren, Czerski, John, Cox, Christopher R., Sarkar, Susanta K.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.08.2018
• Subjects: Collagen - chemistry; Collagen degradation; Electrophoresis, Polyacrylamide Gel; Escherichia coli - genetics; Gelatin - chemistry; Gelatin zymography; Humans; Matrix Metalloproteinase 1 - chemistry; Matrix Metalloproteinase 1 - genetics; Matrix Metalloproteinase 1 - isolation & purification; Protease activity of MMP1; Proteolysis; Recombinant human MMP1; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Protease activity of MMP1; Collagen degradation; Recombinant human MMP1; Gelatin zymography
• ISSN: 1046-5928; 1096-0279
• DOI: 10.1016/j.pep.2018.04.001

MMP1 is an essential enzyme for tissue remodeling both in normal and pathological states. We report a method of purifying activated human MMP1 in E. coli without using urea or 4-Aminophenylmercuric acetate (APMA). Instead, a non-ionic detergent, Triton X-100, was used in the lysis buffer to solubilize MMP1 followed by the protease activities of both trypsin and MMP1 to digest E. coli proteins and activate pro-MMP1. Identity of activated MMP1 was confirmed by Western blot using anti-human MMP1 antibodies, whereas the mass was determined to be 43 kD using matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF-MS). Collagen and gelatin degradation by purified MMP1 were confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) of degraded FITC-labeled type-1 collagen and gelatin zymogram. Broad-spectrum protease activity of purified MMP1 was also confirmed by lysis of native E. coli proteins. Inexpensive high throughput purification of recombinant human MMP1 in E. coli will enable easier MMP1 production for diverse applications.