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Simple assay for proteases based on aggregation of stimulus-responsive polypeptides.

Publication ,  Journal Article
Ghoorchian, A; Chilkoti, A; López, GP
Published in: Analytical chemistry
June 2014

Unregulated changes in protease activity are linked to many diseases including cancer. Fast, accurate, and low-cost assays for detection of these changes are being explored for early diagnosis and monitoring of these diseases and can also be used as platforms for the discovery of new drugs. We report a new methodology for the simple detection and quantification of protease activity in buffer and human serum. The assay is based on recombinant diblock polypeptides that undergo temperature- or salt-triggered micellization in water. The coronae of the micelles are linked to the water-insoluble cores by a peptide substrate that is cleaved in the presence of the target protease. Protease cleavage of the diblock polypeptide triggers the aggregation of the core-forming segment, leading to a change in solution optical density, which can be used to detect the presence of, and to quantify the concentration of, protease. We used matrix metalloproteinase-1 (MMP-1) as a model protease and found peptide aggregation time to be proportional to enzyme concentration over a range from endogenous MMP-1 level in human serum (∼3 ng/mL) to 100 ng/mL (0.15-5 nM) in 40% human serum and 1-100 ng/mL in buffer. The assay does not require any intermediate steps or sophisticated data analysis, and the modular design of the assay system is amenable to straightforward adaptation for the detection of a wide range of proteases.

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Published In

Analytical chemistry

DOI

EISSN

1520-6882

ISSN

0003-2700

Publication Date

June 2014

Volume

86

Issue

12

Start / End Page

6103 / 6110

Related Subject Headings

  • Peptides
  • Molecular Sequence Data
  • Matrix Metalloproteinase 1
  • Mass Spectrometry
  • Humans
  • Analytical Chemistry
  • Amino Acid Sequence
  • 4004 Chemical engineering
  • 3401 Analytical chemistry
  • 3205 Medical biochemistry and metabolomics
 

Citation

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Ghoorchian, A., Chilkoti, A., & López, G. P. (2014). Simple assay for proteases based on aggregation of stimulus-responsive polypeptides. Analytical Chemistry, 86(12), 6103–6110. https://doi.org/10.1021/ac5012574
Ghoorchian, Ali, Ashutosh Chilkoti, and Gabriel P. López. “Simple assay for proteases based on aggregation of stimulus-responsive polypeptides.Analytical Chemistry 86, no. 12 (June 2014): 6103–10. https://doi.org/10.1021/ac5012574.
Ghoorchian A, Chilkoti A, López GP. Simple assay for proteases based on aggregation of stimulus-responsive polypeptides. Analytical chemistry. 2014 Jun;86(12):6103–10.
Ghoorchian, Ali, et al. “Simple assay for proteases based on aggregation of stimulus-responsive polypeptides.Analytical Chemistry, vol. 86, no. 12, June 2014, pp. 6103–10. Epmc, doi:10.1021/ac5012574.
Ghoorchian A, Chilkoti A, López GP. Simple assay for proteases based on aggregation of stimulus-responsive polypeptides. Analytical chemistry. 2014 Jun;86(12):6103–6110.
Journal cover image

Published In

Analytical chemistry

DOI

EISSN

1520-6882

ISSN

0003-2700

Publication Date

June 2014

Volume

86

Issue

12

Start / End Page

6103 / 6110

Related Subject Headings

  • Peptides
  • Molecular Sequence Data
  • Matrix Metalloproteinase 1
  • Mass Spectrometry
  • Humans
  • Analytical Chemistry
  • Amino Acid Sequence
  • 4004 Chemical engineering
  • 3401 Analytical chemistry
  • 3205 Medical biochemistry and metabolomics