so how to cleave off the GST? - (Oct/04/2006 )

molecular cloning book says: add thrombin, enterokinase or Factor Xa (as appropriate for the cleavage site within the fusion protein) can't figure out what that means. and also another question: won't those proteases cleave within my protein too??

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ok i will try to explain it to u ;
i know 3 types to cleave GST ; thrombin and factor xa in one hand and prescission protease in the other hand
the first one its determined from the start ( i mean when preparing ur plasmid with GST-PROTEIN) u add a sequence of recognition by these factors in the junction between gst and ur protein so when adding it later on it will only recognize this sequence spesefically and cut it .
the other type it recognize a specific amino acid residue to cut ( Gln / Gly in this case) so yes u can get ur protein to be cleaved if only it got the same sequence so in this case do sequencing of ur protein first to be sure that its free of this site of cleavage then use this type of protease.

hope this help.

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thank you spanishflower, my GST vector has sequence for thrombin cleavage, but i can't find that of factor Xa... maybe a vector can have only one protease cleavage site?

also another complication: the cleavage site for thrombin is at the C-terminus of GST, but my GST is at the C-terminus of my protein.....so actually it will not cut GST from the protein. what should I do?

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Are you using a pGEX vector?
If so, I'm pretty sure the GST tag is N-terminal of your protein, because I was searching like crazy for a GST fusion vector that yields a C-terminal GST tag but couldn't find one.
Anyway, I strongly recommend moving your insert into the pGEX6P vector, which contains a PreScission cleavage site. That has worked very well for me, whereas Thrombin cleavage on a fusion expressed from pGEX2 worked poorly or not at all. Plus prescission works at 4 deg C.

Good luck!

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hi kathy,
as to whether the thrombin will cleave within your protein you can just check the sequence of your protein on peptide cutter or some other web tool to see if it has a thrombin cleavage site .if it does you will probably have to change vectors. the pgex 6p vectors have prescission protease which is pretty good and supposed to be a lot more specific than thrombin.
i think a vector usually has only one type of protese site in it. but i dont understand. if your vector has a c terminal gst tag then it should also code for a protease site between the tag and the mcs region where you inserted your gene sequence?

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thanx for searching. actually i received this vector from someone. yes it is pGEX, but GST is attached to C-terminus, as requested by authors from the company. it is special request they don;t produce such vectors. but searching in the vector map i can't find any cleavage site between the protein and the GST...

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actually as tested by this peptide cutter

http://kr.expasy.org/tools/peptidecutter/

all enzymes that cut inside GST also cut inside my protein... any other suggestions? maybe other sites?

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Hi Kathy
I am starting working on GST fusion Protein. Can this vector be used for expression on surface of E coli.

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Since the web tool doenst have PreScission, maybe this protease do not cut your protein. PreScission has a larger recognition site, and for that, can be more specific. The recognition is Leu-Glu-Val-Leu-Phe-Gln*Gly-Pro.

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The GST fusion system is recommended for soluble expression of proteins. The only way for directing a protein to the surface is to have another tag such as a peptide leader.

If you really want your protein in the periplasmic you should go for other vector.

cheers

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