Recombinant human TIMP-3 from Escherichia coli: synthesis, refolding, physico-chemical and functional insights.
Matrix metalloproteinases are inhibited by a growing family of specific tissue inhibitors, TIMPs. The cDNA of the third member of the family, TIMP-3, was obtained by using a reverse transcription-polymerase chain reaction (RT-PCR) to amplify the corresponding mRNA from human placenta. Cloning and expression of the TIMP-3 were performed in Escherichia coli as a fusion protein with a 36 amino acid N-tail containing a His cluster. In the host vector system, rhTIMP-3 was stored intracellularly in its denatured, insoluble form in inclusion bodies. Slow dilution of denaturing and reducing agents, from rhTIMP-3 His bound to a metal affinity solid phase, was followed by partial acid removal of the N-tail, which leaves a residue of four amino acids. Circular dichroism, fluorescence and second-derivative UV spectroscopic analyses supported correct refolding of the recombinant and zymography showed inhibition of both MMP-2 and MMP-9 gelatinolytic activities. The role of the C-terminus, which has closer homology with TIMP-2 than TIMP-1, was also investigated: a C-truncated mutant, similarly cloned and expressed in E. coli, shows complete lack of inhibitory activity on MMP-9, still retaining some on MMP-2. The described protein engineering shows high yield of active inhibitor, unglycosylated as in the native form.
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Department of Biological Chemistry, University of Padova, Italy.
Onisto
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M
Grassato
L
L
Caenazzo
C
C
Garbisa
S
S
D000595:Amino Acid Sequence; D018076:DNA, Complementary; D004926:Escherichia coli; D018093:Gelatinases / Q000037:antagonists & inhibitors; D006801:Humans; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9; D061965:Matrix Metalloproteinase Inhibitors; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D008969:Molecular Sequence Data; D011480:Protease Inhibitors / Q000737:chemistry / Q000378:metabolism*; D014176:Protein Biosynthesis; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D019717:Tissue Inhibitor of Metalloproteinase-3; D014407:Tumor Cells, Cultured
D018076:DNA, Complementary; D061965:Matrix Metalloproteinase Inhibitors; D011480:Protease Inhibitors; D011506:Proteins; D011993:Recombinant Fusion Proteins; D019717:Tissue Inhibitor of Metalloproteinase-3; D018093:Gelatinases; D008666:Metalloendopeptidases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9