which protease is good - Molecular Biology

Which protease can be good to place in vector to remove tag from the target protein.
Thrombin, HRV 3C Protease, Enterokinase, Tag is HIS tag and vector and its on c Termianl end.
regards

QUOTE (samita @ Oct 15 2006, 02:42 PM)

Which protease can be good to place in vector to remove tag from the target protein.
Thrombin, HRV 3C Protease, Enterokinase, Tag is HIS tag and vector and its on c Termianl end.
regards

I use thrombin and it works good, but is expensive. The problem is how to determine cleavage of the His tag. There is really no good assay that I know of. 6 or 8 residue differences are really hard to see. How do you plan on determining cleavage?

Currently I have my gene with some solubalilty enhancer tags and Its size is about 17 kd I think if its working on this constrct then i hope it will work on the second constrct as well. I hope it will work if nothing goes wrong. What you think what can be appraoch to see its working or not.
regards

QUOTE (Crystalguy @ Oct 16 2006, 02:01 PM)

QUOTE (samita @ Oct 15 2006, 02:42 PM)

Which protease can be good to place in vector to remove tag from the target protein.
Thrombin, HRV 3C Protease, Enterokinase, Tag is HIS tag and vector and its on c Termianl end.
regards

I use thrombin and it works good, but is expensive. The problem is how to determine cleavage of the His tag. There is really no good assay that I know of. 6 or 8 residue differences are really hard to see. How do you plan on determining cleavage?

QUOTE (samita @ Oct 17 2006, 07:40 AM)

Currently I have my gene with some solubalilty enhancer tags and Its size is about 17 kd I think if its working on this constrct then i hope it will work on the second constrct as well. I hope it will work if nothing goes wrong. What you think what can be appraoch to see its working or not.
regards

QUOTE (Crystalguy @ Oct 16 2006, 02:01 PM)

QUOTE (samita @ Oct 15 2006, 02:42 PM)

Which protease can be good to place in vector to remove tag from the target protein.
Thrombin, HRV 3C Protease, Enterokinase, Tag is HIS tag and vector and its on c Termianl end.
regards

I use thrombin and it works good, but is expensive. The problem is how to determine cleavage of the His tag. There is really no good assay that I know of. 6 or 8 residue differences are really hard to see. How do you plan on determining cleavage?

What vector are you using? I am assuming the solubility enhancers will be cut off during cleavage, so you will be able to see a size shift on a gel.

I am using the pET32A. But today i have a new strategy I will cut my pET32a+ vector at MSC1 site and clone between MSC1 and XHO1 site then after that i will be able to use Histidine tag at C-tail from pET32 a+. But i have 12 kd size protein fragement from vector sequece in addition to my protein sequecne before MSC1 site. To remove this 12kd or His tags from my protein sequence i was asked to use 2 different protease. Is it ok or use the one protease for the both HIS and Vector coded protein sequece. I am planning to use HRV 3C Protease and enterokinase. Enterkinase at C, terminus and Thrombin at the N-terminus. What you think.
regards

QUOTE (Crystalguy @ Oct 17 2006, 11:18 AM)

QUOTE (samita @ Oct 17 2006, 07:40 AM)

Currently I have my gene with some solubalilty enhancer tags and Its size is about 17 kd I think if its working on this constrct then i hope it will work on the second constrct as well. I hope it will work if nothing goes wrong. What you think what can be appraoch to see its working or not.
regards

QUOTE (Crystalguy @ Oct 16 2006, 02:01 PM)

QUOTE (samita @ Oct 15 2006, 02:42 PM)

Which protease can be good to place in vector to remove tag from the target protein.
Thrombin, HRV 3C Protease, Enterokinase, Tag is HIS tag and vector and its on c Termianl end.
regards

I use thrombin and it works good, but is expensive. The problem is how to determine cleavage of the His tag. There is really no good assay that I know of. 6 or 8 residue differences are really hard to see. How do you plan on determining cleavage?

What vector are you using? I am assuming the solubility enhancers will be cut off during cleavage, so you will be able to see a size shift on a gel.

QUOTE (samita @ Oct 17 2006, 06:08 PM)

I am using the pET32A. But today i have a new strategy I will cut my pET32a+ vector at MSC1 site and clone between MSC1 and XHO1 site then after that i will be able to use Histidine tag at C-tail from pET32 a+. But i have 12 kd size protein fragement from vector sequece in addition to my protein sequecne before MSC1 site. To remove this 12kd or His tags from my protein sequence i was asked to use 2 different protease. Is it ok or use the one protease for the both HIS and Vector coded protein sequece. I am planning to use HRV 3C Protease and enterokinase. Enterkinase at C, terminus and Thrombin at the N-terminus. What you think.
regards

QUOTE (Crystalguy @ Oct 17 2006, 11:18 AM)

QUOTE (samita @ Oct 17 2006, 07:40 AM)

Currently I have my gene with some solubalilty enhancer tags and Its size is about 17 kd I think if its working on this constrct then i hope it will work on the second constrct as well. I hope it will work if nothing goes wrong. What you think what can be appraoch to see its working or not.
regards

QUOTE (Crystalguy @ Oct 16 2006, 02:01 PM)

QUOTE (samita @ Oct 15 2006, 02:42 PM)

Which protease can be good to place in vector to remove tag from the target protein.
Thrombin, HRV 3C Protease, Enterokinase, Tag is HIS tag and vector and its on c Termianl end.
regards

I use thrombin and it works good, but is expensive. The problem is how to determine cleavage of the His tag. There is really no good assay that I know of. 6 or 8 residue differences are really hard to see. How do you plan on determining cleavage?

What vector are you using? I am assuming the solubility enhancers will be cut off during cleavage, so you will be able to see a size shift on a gel.

I looked at the pET32A vector map. If you cut with MscI and XhoI, you will remove both the thrombin and enterokinase site. If I was cloning into pET32a, I would use NcoI and XhoI (NcoI to limit the amount of extra residues left over after cleavage). I would use the N-terminal His Tag and cut with enterokinase only. You should be able to see a size difference between the cut and uncut. I put three stops at the end of all my genes, so I always use the N-terminal His tag. The C-terminal tag never gets expressed. You can also cut on the column, but I've had limited success with it. You can then do ion exchange and gel filtration to finish the purification.
I have also used pMAL successfully to help keep proteins soluble. MBP is the selection marker and it is 42 kDa. It's really easy to see when it's been cut. Hope this helps.

Actually I am currently expressing my gene in pET 32A with Ecor5 and HindIII site in whihc all protease enzmyme site and His tag are at the N-terminal tag. But my protein is forming inclusion body, Tag is two big and 17 KD and my protein is about 24kd. and Low binding to affininty column (Cobalt). But i have reviewed some papers and some thing similar proein expresed in pET21 vector and they are using C-terminal His Tag but i donot have this vector so to make pET32 A+ to pET21 vector to access c-terminal His tag i am cloning in MSC1 and XHOI site. After this i will be able to use C-teminal His Taq and addition 12kd prtein sequecen at start of the vector i will remove by adding the protease site. I try pMAL and after getting colony i put it on shaker for overnight culture and it does not grow at all. I try pAML with TB1 cell but now i am thinking to try it with BL21 cell. I have membrane protein. please give comments
regards

QUOTE (Crystalguy @ Oct 17 2006, 04:25 PM)

QUOTE (samita @ Oct 17 2006, 06:08 PM)

I am using the pET32A. But today i have a new strategy I will cut my pET32a+ vector at MSC1 site and clone between MSC1 and XHO1 site then after that i will be able to use Histidine tag at C-tail from pET32 a+. But i have 12 kd size protein fragement from vector sequece in addition to my protein sequecne before MSC1 site. To remove this 12kd or His tags from my protein sequence i was asked to use 2 different protease. Is it ok or use the one protease for the both HIS and Vector coded protein sequece. I am planning to use HRV 3C Protease and enterokinase. Enterkinase at C, terminus and Thrombin at the N-terminus. What you think.
regards

QUOTE (Crystalguy @ Oct 17 2006, 11:18 AM)

QUOTE (samita @ Oct 17 2006, 07:40 AM)

Currently I have my gene with some solubalilty enhancer tags and Its size is about 17 kd I think if its working on this constrct then i hope it will work on the second constrct as well. I hope it will work if nothing goes wrong. What you think what can be appraoch to see its working or not.
regards

QUOTE (Crystalguy @ Oct 16 2006, 02:01 PM)

QUOTE (samita @ Oct 15 2006, 02:42 PM)

Which protease can be good to place in vector to remove tag from the target protein.
Thrombin, HRV 3C Protease, Enterokinase, Tag is HIS tag and vector and its on c Termianl end.
regards

I use thrombin and it works good, but is expensive. The problem is how to determine cleavage of the His tag. There is really no good assay that I know of. 6 or 8 residue differences are really hard to see. How do you plan on determining cleavage?

What vector are you using? I am assuming the solubility enhancers will be cut off during cleavage, so you will be able to see a size shift on a gel.

I looked at the pET32A vector map. If you cut with MscI and XhoI, you will remove both the thrombin and enterokinase site. If I was cloning into pET32a, I would use NcoI and XhoI (NcoI to limit the amount of extra residues left over after cleavage). I would use the N-terminal His Tag and cut with enterokinase only. You should be able to see a size difference between the cut and uncut. I put three stops at the end of all my genes, so I always use the N-terminal His tag. The C-terminal tag never gets expressed. You can also cut on the column, but I've had limited success with it. You can then do ion exchange and gel filtration to finish the purification.
I have also used pMAL successfully to help keep proteins soluble. MBP is the selection marker and it is 42 kDa. It's really easy to see when it's been cut. Hope this helps.

QUOTE (samita @ Oct 18 2006, 04:38 AM)

Actually I am currently expressing my gene in pET 32A with Ecor5 and HindIII site in whihc all protease enzmyme site and His tag are at the N-terminal tag. But my protein is forming inclusion body, Tag is two big and 17 KD and my protein is about 24kd. and Low binding to affininty column (Cobalt). But i have reviewed some papers and some thing similar proein expresed in pET21 vector and they are using C-terminal His Tag but i donot have this vector so to make pET32 A+ to pET21 vector to access c-terminal His tag i am cloning in MSC1 and XHOI site. After this i will be able to use C-teminal His Taq and addition 12kd prtein sequecen at start of the vector i will remove by adding the protease site. I try pMAL and after getting colony i put it on shaker for overnight culture and it does not grow at all. I try pAML with TB1 cell but now i am thinking to try it with BL21 cell. I have membrane protein. please give comments
regards

QUOTE (Crystalguy @ Oct 17 2006, 04:25 PM)

QUOTE (samita @ Oct 17 2006, 06:08 PM)

I am using the pET32A. But today i have a new strategy I will cut my pET32a+ vector at MSC1 site and clone between MSC1 and XHO1 site then after that i will be able to use Histidine tag at C-tail from pET32 a+. But i have 12 kd size protein fragement from vector sequece in addition to my protein sequecne before MSC1 site. To remove this 12kd or His tags from my protein sequence i was asked to use 2 different protease. Is it ok or use the one protease for the both HIS and Vector coded protein sequece. I am planning to use HRV 3C Protease and enterokinase. Enterkinase at C, terminus and Thrombin at the N-terminus. What you think.
regards

QUOTE (Crystalguy @ Oct 17 2006, 11:18 AM)

QUOTE (samita @ Oct 17 2006, 07:40 AM)

Currently I have my gene with some solubalilty enhancer tags and Its size is about 17 kd I think if its working on this constrct then i hope it will work on the second constrct as well. I hope it will work if nothing goes wrong. What you think what can be appraoch to see its working or not.
regards

QUOTE (Crystalguy @ Oct 16 2006, 02:01 PM)

QUOTE (samita @ Oct 15 2006, 02:42 PM)

Which protease can be good to place in vector to remove tag from the target protein.
Thrombin, HRV 3C Protease, Enterokinase, Tag is HIS tag and vector and its on c Termianl end.
regards

I use thrombin and it works good, but is expensive. The problem is how to determine cleavage of the His tag. There is really no good assay that I know of. 6 or 8 residue differences are really hard to see. How do you plan on determining cleavage?

What vector are you using? I am assuming the solubility enhancers will be cut off during cleavage, so you will be able to see a size shift on a gel.

I looked at the pET32A vector map. If you cut with MscI and XhoI, you will remove both the thrombin and enterokinase site. If I was cloning into pET32a, I would use NcoI and XhoI (NcoI to limit the amount of extra residues left over after cleavage). I would use the N-terminal His Tag and cut with enterokinase only. You should be able to see a size difference between the cut and uncut. I put three stops at the end of all my genes, so I always use the N-terminal His tag. The C-terminal tag never gets expressed. You can also cut on the column, but I've had limited success with it. You can then do ion exchange and gel filtration to finish the purification.
I have also used pMAL successfully to help keep proteins soluble. MBP is the selection marker and it is 42 kDa. It's really easy to see when it's been cut. Hope this helps.

Membrane protein puts a whole new spin on things. The cause of your problem could be in your lysis/purification protocol. What kind of detergent are you using? I use 2% DDM and 0.5M Sucrose for my membrane protein preps. I can send you the exact protocol if you want. I have had good success using it.

which protease is good - (Oct/15/2006 )