Purification of Human Matrilysin Produced inEscherichia coliand Characterization Using a New Optimized Fluorogenic Peptide Substrate

Welch, Anthony; Holman, Christopher M.; Browner, Michelle F.; Gehring, Michael R.; Kan, Chen‐Chen; Wart, Harold E. Van

doi:10.1006/abbi.1995.9929

Cited by 38 publications

(24 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The nucleotide sequence coding for residues Leu 20‐Asn 274 coded for by cDNA (Freije et al , 1994) of the catalytic domain of procollagenase‐3 was amplified from the cDNA. The amplified product was isolated and ligated into KpnI/EcoRI digested pUbPMt (Welch et al , 1995). An N‐terminal ubiquitin‐procollagenase‐3 fusion protein was produced in E. coli by expression from the pHC3x.2 plasmid in BL21 (DE3) cells.…”

Section: Methodsmentioning

confidence: 99%

Ro 32‐3555, an orally active collagenase inhibitor, prevents cartilage breakdown in vitro and in vivo

Lewis

Bishop

Bottomley

et al. 1997

British J Pharmacology

132

View full text Add to dashboard Cite

show abstract

Section: Methodsmentioning

confidence: 99%

Ro 32‐3555, an orally active collagenase inhibitor, prevents cartilage breakdown in vitro and in vivo

Lewis

Bishop

Bottomley

et al. 1997

British J Pharmacology

132

View full text Add to dashboard Cite

show abstract

“…In addition, there are a number of more selective fluorogenic peptide substrates for proteases, including some that can be cleaved by MMPs. One such fluorogenic peptide was designed for cleavage by MMP-7 [10]. These kinds of peptide-based substrates serve to assay MMP activities in vitro, but have not been used as in vivo reporters of enzymic activity.…”

Section: Introductionmentioning

confidence: 99%

Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity

McIntyre

Fingleton

Wells

et al. 2004

Biochemical Journal

145

View full text Add to dashboard Cite

The present study describes the in vivo detection and imaging of tumour-associated MMP-7 (matrix metalloproteinase-7 or matrilysin) activity using a novel polymer-based fluorogenic substrate PB-M7VIS, which serves as a selective 'proteolytic beacon' (PB) for this metalloproteinase. PB-M7VIS is built on a PAMAM (polyamido amino) dendrimer core of 14.2 kDa, covalently coupled with an Fl (fluorescein)-labelled peptide Fl(AHX)RPLALWRS(AHX)C (where AHX stands for aminohexanoic acid) and with TMR (tetramethylrhodamine). PB-M7VIS is efficiently and selectively cleaved by MMP-7 with a k (cat)/ K (m) value of 1.9x10(5) M(-1).s(-1) as measured by the rate of increase in Fl fluorescence (up to 17-fold for the cleavage of an optimized PB-M7VIS) with minimal change in the TMR fluorescence. The K (m) value for PB-M7VIS is approx. 0.5 microM, which is approx. two orders of magnitude lower when compared with that for an analogous soluble peptide, indicating efficient interaction of MMP-7 with the synthetic polymeric substrate. With MMP-2 or -3, the k (cat)/ K (m) value for PB-M7VIS is approx. 56- or 13-fold lower respectively, when compared with MMP-7. In PB-M7VIS, Fl(AHX)RPLALWRS(AHX)C is a selective optical sensor of MMP-7 activity and TMR serves to detect both the uncleaved and cleaved reagents. Each of these can be visualized as subcutaneous fluorescent phantoms in a mouse and optically discriminated based on the ratio of green/red (Fl/TMR) fluorescence. The in vivo specificity of PB-M7VIS was tested in a mouse xenograft model. Intravenous administration of PB-M7VIS gave significantly enhanced Fl fluorescence from MMP-7-positive tumours, but not from control tumours ( P <0.0001), both originally derived from SW480 human colon cancer cells. Prior systemic treatment of the tumour-bearing mice with an MMP inhibitor BB-94 ([4-( N -hydroxyamino)-2 R -isobutyl-3 S -(thienylthiomethyl)-succinyl]-L-phenylalanine- N -methylamide), markedly decreased the Fl fluorescence over the MMP-7-positive tumour by approx. 60%. Thus PB-M7VIS functions as a PB for in vivo detection of MMP-7 activity that serves to light this optical beacon and is, therefore, a selective in vivo optical molecular imaging contrast reagent.

show abstract

“…Previous literature reports the K m of unmodifi ed PA substrate as 26 µM (Welch et al 1995). Thus, at substrate concentrations below this value, PA cleavage is expected to be independent of MMP-7 concentration.…”

Section: Qd-pa Construct Synthesis and Activitymentioning

confidence: 90%

Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

Giorgio¹

2008

IJN

View full text Add to dashboard Cite

Abstract:The development and in vitro performance of a modular nanoscale system capable of specifi c structural modifi cation by enzymatic activity is described in this work. Due to its small physical size and adaptable characteristics, this system has the potential for utilization in targeted delivery systems and biosensing. Nanoparticle probes were synthesized containing two distinct fl uorescent species including a quantum dot base particle and fl uorescently labeled cleavable peptide substrate. Activity of these probes was monitored by gel electrophoresis with quantitative cleavage measurements made by fl uorometric analysis. The model proximityactivated nanoparticles studied here exhibit signifi cant susceptibility to cleavage by matrix metalloprotease-7 (MMP-7) at physiologically relevant concentrations, with nearly complete cleavage of available substrate molecules after 24 hours. This response is specifi c to MMP-7 enzyme activity, as cleavage is completely inhibited with the addition of EDTA. Utilization of enzyme-specifi c modifi cation is a sensitive approach with broad applications for targeted therapeutics and biosensing. The versatility of this nanoparticle system is highlighted in its modular design, as it has the capability to integrate characteristics for detection, biosensing, targeting, and payload delivery into a single, multifunctional nanoparticle structure.

show abstract

Purification of Human Matrilysin Produced inEscherichia coliand Characterization Using a New Optimized Fluorogenic Peptide Substrate

Cited by 38 publications

References 0 publications

Ro 32‐3555, an orally active collagenase inhibitor, prevents cartilage breakdown in vitro and in vivo

Ro 32‐3555, an orally active collagenase inhibitor, prevents cartilage breakdown in vitro and in vivo

Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity

Proximity-activated nanoparticles: in vitro performance of specific structural modification by enzymatic cleavage

Contact Info

Product

Resources

About