IGEM:Imperial/2010/Fast Response module/Target Proteases
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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:
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
References:
Monitoring regulated protein-protein interactions using split TEV
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