Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Insertion of an elastase-binding loop into interleukin-1 beta.

A J Wolfson1, M Kanaoka, F T Lau

  • 1Brandeis University, Rosentiel Basic Medical Research Center, Waltham, MA 02254.

Protein Engineering
|February 1, 1991
PubMed
Summary
This summary is machine-generated.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Serum thymus and activation-regulated chemokine (TARC/CCL17) may be useful to predict the disease activity in patients with bullous pemphigoid.

Journal of the European Academy of Dermatology and Venereology : JEADV·2020
Same author

Health-related quality of life in patients with lower rectal cancer after sphincter-saving surgery: a prospective 6-month follow-up study.

European journal of cancer care·2015
Same author

Preservation of forelimb function by UPF1 gene therapy in a rat model of TDP-43-induced motor paralysis.

Gene therapy·2014
Same author

All-atom empirical potential for molecular modeling and dynamics studies of proteins.

The journal of physical chemistry. B·2014
Same author

Latrepirdine stimulates autophagy and reduces accumulation of α-synuclein in cells and in mouse brain.

Molecular psychiatry·2012
Same author

Latrepirdine improves cognition and arrests progression of neuropathology in an Alzheimer's mouse model.

Molecular psychiatry·2012
Same journal

Structure of a human Rhinovirus complexed with its receptor molecule.

Protein engineering·2024
Same journal

pH-responsive polymer-assisted refolding of urea- and organic solvent-denatured alpha-chymotrypsin.

Protein engineering·2004
Same journal

Evaluation of different linker regions for multimerization and coupling chemistry for immobilization of a proteinaceous affinity ligand.

Protein engineering·2004
Same journal

Recombinant porcine intestinal carboxylesterase: cloning from the pig liver esterase gene by site-directed mutagenesis, functional expression and characterization.

Protein engineering·2004
Same journal

Periplasmic expression of human growth hormone via plasmid vectors containing the lambdaPL promoter: use of HPLC for product quantification.

Protein engineering·2004
Same journal

Shift of fibril-forming ability of the designed alpha-helical coiled-coil peptides into the physiological pH region.

Protein engineering·2004
See all related articles

Researchers engineered a new protein by inserting an alpha 1-antitrypsin sequence into interleukin-1 beta. This mutant protein is cleaved by specific proteases and inhibits elastase, showing functional protease-binding activity in a new context.

Area of Science:

  • Protein engineering and molecular biology
  • Enzymology and protease inhibition
  • Biochemistry and structural biology

Background:

  • Alpha 1-antitrypsin is a key inhibitor of serine proteases.
  • Interleukin-1 beta is a cytokine involved in inflammation and cellular processes.
  • Modifying protein structures can alter their functional properties.

Purpose of the Study:

  • To investigate if the protease-binding sequence of alpha 1-antitrypsin retains activity when inserted into a different protein.
  • To determine if the engineered interleukin-1 beta mutant can be cleaved by proteases.
  • To assess the potential of interleukin-1 beta as a scaffold for therapeutic agents.

Main Methods:

  • Site-directed mutagenesis to insert the EAIPMSIPPE sequence from alpha 1-antitrypsin into interleukin-1 beta.

Related Experiment Videos

  • Enzymatic cleavage assays using elastase, chymotrypsin, and trypsin.
  • Circular dichroism (CD) spectroscopy to assess protein structure.
  • Determination of inhibition constants (KI) for elastase inhibition.

    • Main Results:

    • The engineered mutant protein was specifically cleaved by elastase and chymotrypsin at the inserted sequence, unlike wild-type interleukin-1 beta.
    • The mutant protein demonstrated inhibitory activity against elastase with a KI of approximately 30 microM.
    • Circular dichroism showed no significant structural differences between the mutant and wild-type proteins.
    • Cleavage by elastase occurred specifically between Methionine and Serine in the inserted loop.

    Conclusions:

    • The protease-binding sequence of alpha 1-antitrypsin is functional and recognized within the context of the interleukin-1 beta structure.
    • This engineered hybrid protein exhibits specific protease inhibition.
    • Interleukin-1 beta can serve as a viable delivery system for therapeutic agents due to its cellular uptake properties.