Production of a bioactive recombinant chicken matrix metalloproteinase-11 peptide in Escherichia coli.
Matrix metalloproteinase-11 (MMP-11) is known to be highly expressed in metastatic and most invasive forms of tumors. Being selectively expressed in tumor tissues, MMP-11 is a promising target for immunotherapy against tumors. Here, we report the production of a thioredoxin-tagged bioactive recombinant chicken MMP-11 (cMMP-11) peptide excluding the secretory signal and propeptide in Escherichia coli T7 Express lysY using pET32b(+) vector. High-level expression and purification of the bioactive peptide were achieved by induction with 1.0 mM isopropyl-β-d-thiogalactopyranoside for 4 H at 37 °C followed by affinity chromatography under denaturing condition and slow dialysis. The recombinant peptide exhibited both caseinolytic and gelatinase activities without requiring activation by 4-aminophenylmercuric acetate. The antisera raised against the peptide in rabbits showed a strong reaction with the whole recombinant peptide as well as 37 kDa cMMP-11 mature peptide and cross-reactivity with a 43 kDa protein in murine breast tumor of 4T1 origin in Western blot analysis. The 43 kDa protein in the tumor homogenate showed immunoreactivity with a monoclonal antibody against human MMP-11, suggesting it to be murine MMP-11 having cross-reactivity with the antisera raised against cMMP-11 peptide. Altogether, the study characterized the production of a bioactive and immunogenic recombinant cMMP-11 peptide in E. coli.
Division of Animal Biochemistry, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India.
Rajani Kumar
RK
Paul
Division of Animal Biochemistry, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India.
Mukesh
M
Kumar
Division of Animal Biochemistry, ICAR-Indian Veterinary Research Institute, Bareilly, Uttar Pradesh, India.
Meena
M
Kataria
D000818:Animals; D002645:Chickens; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D053507:Matrix Metalloproteinase 11 / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D011994:Recombinant Proteins; D053507:Matrix Metalloproteinase 11
MMP-11; chicken; prokaryotic expression; purification; zymography