Presence of an N-Terminal Polyhistidine Tag Facilitates Stable Expression of an Otherwise Unstable N-Terminal Domain of Mouse Tissue Inhibitor of Metalloproteinase-1 inEscherichia coli

1998 ◽  
Vol 13 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Shyamala S. Rajan ◽  
Henry Lackland ◽  
Stanley Stein ◽  
David T. Denhardt
Keyword(s):  
Stable Expression ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Mouse Tissue ◽  
Inescherichia Coli ◽  
Tissue Inhibitor ◽  
Terminal Domain

Cloning and sequencing of the cDNA encoding a mouse tissue inhibitor of metalloproteinase-2

Gene ◽  
1992 ◽  
Vol 114 (2) ◽  
pp. 291-292 ◽  
Author(s):  
Satoru Shimizu ◽  
Karim Malik ◽  
Hiroshi Sejima ◽  
Jun-ichi Kischi ◽  
Taro Hayakawa ◽  
...  
Keyword(s):  
Tissue Inhibitor Of Metalloproteinase ◽  
Mouse Tissue ◽  
Cloning And Sequencing ◽  
Tissue Inhibitor

scholarly journals Engineering N-terminal domain of tissue inhibitor of metalloproteinase (TIMP)-3 to be a better inhibitor against tumour necrosis factor-α-converting enzyme

2002 ◽  
Vol 364 (1) ◽  
pp. 227-234 ◽  
Author(s):  
Meng-Huee LEE ◽  
Vandana VERMA ◽  
Klaus MASKOS ◽  
Deepa NATH ◽  
Vera KNÄUPER ◽  
...  
Keyword(s):  
Full Length ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Binding Affinities ◽  
Converting Enzyme ◽  
Wild Type ◽  
Tissue Inhibitor ◽  
Terminal Domain ◽  
Tnf Α ◽  
Factor Α ◽  
Necrosis Factor

We previously reported that full-length tissue inhibitor of metalloproteinase-3 (TIMP-3) and its N-terminal domain form (N-TIMP-3) displayed equal binding affinity for tissue necrosis factor-α (TNF-α)-converting enzyme (TACE). Based on the computer graphic of TACE docked with a TIMP-3 model, we created a number of N-TIMP-3 mutants that showed significant improvement in TACE inhibition. Our strategy was to select those N-TIMP-3 residues that were believed to be in actual contact with the active-site pockets of TACE and mutate them to amino acids of a better-fitting nature. The activities of these mutants were examined by measuring their binding affinities (Kappi) and association rates (kon) against TACE. Nearly all mutants at position Thr-2 exhibited slightly impaired affinity as well as association rate constants. On the other hand, some Ser-4 mutants displayed a remarkable increase in their binding tightness with TACE. In fact, the binding affinities of several mutants were less than 60pM, beyond the sensitivity limits of fluorimetric assays. Further studies on cell-based processing of pro-TNF-α demonstrated that wild-type N-TIMP-3 and one of its tight-binding mutants, Ser-4Met, were capable of inhibiting the proteolytic shedding of TNF-α. Furthermore, the Ser-4Met mutant was also significantly more active (P<0.05) than the wild-type N-TIMP-3 in its cellular inhibition. Comparison of N-TIMP-3 and full-length TIMP-3 revealed that, despite their identical TACE-interaction kinetics, the latter was nearly 10 times more efficient in the inhibition of TNF-α shedding, with concomitant implications for the importance of the TIMP-3 C-terminal domain in vivo.

scholarly journals The C-terminal domain of 72 kDa gelatinase A is not required for catalysis, but is essential for membrane activation and modulates interactions with tissue inhibitors of metalloproteinases

1992 ◽  
Vol 283 (3) ◽  
pp. 637-641 ◽  
Author(s):  
G Murphy ◽  
F Willenbrock ◽  
R V Ward ◽  
M I Cockett ◽  
D Eaton ◽  
...  
Keyword(s):  
Kinetic Studies ◽  
Full Length ◽  
Tissue Inhibitor Of Metalloproteinase ◽  
Tissue Inhibitors Of Metalloproteinases ◽  
Gelatinase A ◽  
Truncated Form ◽  
Tissue Inhibitor ◽  
Tissue Inhibitors ◽  
Terminal Domain

Recombinant 72 kDa gelatinase A and a truncated form lacking the C-terminal domain were shown to be activated by organomercurials and to possess similar activities towards a number of substrates. The truncated proenzyme differed from the full-length gelatinase in that it could not be activated by a membrane activator and did not bind tissue inhibitor of metalloproteinase (TIMP)-2. Kinetic studies also showed that the inhibition of the activated truncated enzyme, by both TIMP-1 and TIMP-2, was considerably decreased compared with the full-length enzyme. We conclude that the C-terminal domain plays an important role in the regulation of gelatinase A by a potential physiological activator and inhibitors.

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