IGEM:Imperial/2010/Fast Response module/Target Proteases

From OpenWetWare

Jump to: navigation, search

Contents

Target Proteases

Up to now we came up with 2 candidate proteins that we could use in our system for the cleavage of the polypeptide:

  • HIV-1 protease
  • TEV

HIV-1 protease

HIV-1 could be a potential candidate as extensive research on this protein already exists. The following characteristics make it a good candidate:

  • with codon optimization the expression of this protein can reach up to 8-10% of the total protein expression
  • the protein can be expressed in E.coli in its active form
  • is produced as a fussion protein which helps to reduce cytotoxicity
  • the expression of fussion protein could confer an advantage of phorylation regulated fussion partner
  • dimer of 99 amino acids which allows for fast transcription-translation
  • optimun pH 6
  • Kcat/Km = 17 per mM per sec

References:

Kinetic Characterization and Cross-Resistance Patterns Of HIV-1 Protease Mutants Selected under Drug Pressure

High-level production of active HIV-1 protease in Escherichia coli.

TEV protease

  • Naturally a polyprotein cutting protease
  • Expression in E.Coli is made efficient by codon optimisation and inducing expression of additional tRNAs
  • pH 7.6
  • Optimum temp. 37˚C works well in 20-25˚C too.
  • this enzyme is extensively used in protein engineering but there are some concerns whether this enzymes is functional inside the cell
  • high specificity
  • No data found on the Michaelis constant nor catalytic constant


Split protease

  • N-TEV(1-118)/C-TEV(119-242)
  • N-tev(1-70)/C-TEV(61-242)
  • 30-40% activity
  • if substrate immobilised (bound) to protease rather than free in cytoplasm-->increased activity


Engineered TEV cleavage site between two fluorescent proteins to assay for TEV activity:

Image:Linkerfret.JPG Taken from ref

References:

Monitoring regulated protein-protein interactions using split TEV

Processive Degradation of Nascent Polypeptides,Triggered by Tandem AGA Codons, Limits the Accumulation of Recombinant Tobacco Etch Virus Protease in Escherichia coli BL21

Suggestions

if you have any constructive comments please add here using the edit function

  • Chris D Hirst 12:08, 15 July 2010 (EDT): What would be the potential method of activation of these proteases?

the activation would be accomplished by bringing together the two split domains of the protease by the association of the binding partners!

  • Chris D Hirst 05:13, 16 July 2010 (EDT): I'm aware that one of these proteases on the registry has already been split for use, is there any data on how effective that worked? Is there any data on plitting any of the others so we would have an indication of where to split them?
Personal tools