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

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:

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-TEV91-1180/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

References:

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?