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Factor XA secondary cleavage sites


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#1 pET

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Posted 10 August 2009 - 04:03 AM

Dear all
I want to do expression of my protein in E.coli with a His Tag, to be able to cleave the tag afterwards I plan to insert a Factor XA cleavage site. At New England Biolabs they remark that a secondary cleavage site for Factor XA is Gly-Arg primarily when proteins are unfolded. I wonder now if anyone ever experienced that effect because Gly-Arg is a rather common motive in my opinion, and Factor XA a rather common used protease, also I did not find an indication for the secondary cleavage anywhere else.
Should I worry using the Factor XA in constructs with Gly-Arg now ?
pET

#2 T C

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Posted 13 August 2009 - 11:58 PM

Hey,

Don't know about this but another strategy involves engineering a Caspase site, which enables you to cleave the tag efficiently at 4 degrees.

Best,
TC

Dear all
I want to do expression of my protein in E.coli with a His Tag, to be able to cleave the tag afterwards I plan to insert a Factor XA cleavage site. At New England Biolabs they remark that a secondary cleavage site for Factor XA is Gly-Arg primarily when proteins are unfolded. I wonder now if anyone ever experienced that effect because Gly-Arg is a rather common motive in my opinion, and Factor XA a rather common used protease, also I did not find an indication for the secondary cleavage anywhere else.
Should I worry using the Factor XA in constructs with Gly-Arg now ?
pET






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