how to insert a thrombin cleavage site - ....into my vector (Mar/13/2008 )
Hi there!
I'd like to introduce into my expression vector a thrombin cleavage site. Because its a special case I can not use a ready to use plasmid where a thrombin cleavage site already exists. While thrombin recognizes "Leu-Val-Pro-Arg-Gly-Ser" I wonder whether I need a spacer before and after the sequence. If so would I need only one spacer if I have a His6-tag at the other side?
Tat,
According to Sigma (http://www.sigmaaldrich.com/Area_of_Interest/Biochemicals/Enzyme_Explorer/Analytical_Enzymes/Thrombins.html)
"The optimal cleavage sites for thrombin have been determined to be 1) A-B-Pro-Arg-||-X-Y where A and B are hydrophobic amino acids and X and Y are nonacidic amino acids and 2) Gly-Arg-||-Gly."
It doesn't look like you need a spacer if you are going to digest it later with thrombin.
I'd like to introduce into my expression vector a thrombin cleavage site. Because its a special case I can not use a ready to use plasmid where a thrombin cleavage site already exists. While thrombin recognizes "Leu-Val-Pro-Arg-Gly-Ser" I wonder whether I need a spacer before and after the sequence. If so would I need only one spacer if I have a His6-tag at the other side?
My suggestion is a straightforward cloning exercise. Synthesise the sequence you need, with either the specific RE sites at each end, or else with 5-10 bp overlap which you then digest with the required enzyme. Clone as normal.
I'd like to introduce into my expression vector a thrombin cleavage site. Because its a special case I can not use a ready to use plasmid where a thrombin cleavage site already exists. While thrombin recognizes "Leu-Val-Pro-Arg-Gly-Ser" I wonder whether I need a spacer before and after the sequence. If so would I need only one spacer if I have a His6-tag at the other side?
My suggestion is a straightforward cloning exercise. Synthesise the sequence you need, with either the specific RE sites at each end, or else with 5-10 bp overlap which you then digest with the required enzyme. Clone as normal.
We use LVPRGS and it works very well. This sequence can be inserted into your vector using Quickchange XL kit if you want to be fancy. Whether or not you need a spacer is going to be protein dependant...if you can throw two AA in there after your protein and before the thrombin site just to be safe.