Dear all,
I would like to know if any of you express and purify the protease enzyme factor Xa in your lab. I have a protein construct with a factor Xa cleavage site to remove the tag. I would have to use a lot of factor Xa (supplied by NEB) to get my target protein cleaved off from the tag.
My protein of interest is a short peptide nearly 4.5 kDa size and tag is the humongous MBP which is over 40 kDa. So, in terms of cost it is not a viable option to use the NEB factor Xa as it would cost us thousands of US$. It would be awesome if any of you guys have a protein expression plasmid for factor Xa.
I will be very grateful if you could lend me a tiny aliquot of your plasmid. Please get in touch with me. Thank you very much.
Sunny
Hi Sunny- I am sure you will be disappointed because FXa is not that specific. Sigma has bovine FXa for sale at $120 for 50 ug.
FXa is a bit more specific than thrombin, but not by much.
If your peptide sequence will tolerate it, then thrombin is sometimes useful and much more available.
e.g. 1. Liu D, Xu R, Dutta K, & Cowburn D (2008) N-terminal cysteinyl proteins can be prepared using thrombin cleavage. FEBS Lett 582(7):1163-1167.
Thank you Steingrimur and David for your replies. I appreciate it very much.
So, David shall I then change the factor Xa cleavage site to that of thrombin in this pMAL-c5X expression vector ensuring everything is in the correct reading frame?
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