co-expression vectors - need info.. (May/24/2011 )
Hey I want to co-express two genes in the same bacteria ..
For it the ori of replication shld be diff and compatible...can pUC vectors (with COlE1) be used for co-expression with pET vectors....I am confused about the ori of replication in pET...some say it has both f1 and COLE1 and in some like in pET21 its F1...which one is used for expression? Also what is the ori of replication in pET28a vectors..I already have one of them cloned in it....
expression of one the two proteins I am interested in depends on the rate of expression of another...so should I clone the highly soluble and well expressing protein in low copy number plasmid and the low expressing one in high copy number and then try to co-express both?
As I am not that good with cloning...need help!!!
thanxz
pUC vectors and pET vector poses the same ColE1-like origin and are therefore not compatible (although they can be maintained by using selection pressure when both plasmids contain different resistance genes). The f1 origin is only used for the generation of single stranded DNA and is not able to replicate the plasmid in bacteria!!!
The copy number of your vector is a crucial parameter in protein expression. If you use the T7 system for expression of protein even high protein yields can be achieved with low copy numbers ...if you use promoters like lac or tac you can use higher copy numbers. It always depends on your protein and what is your ultimate goal ...high content of soluble protein or insoluble protein in form of inclusion bodies ...or something else.
Maybe we can help you if you will describe your experiment in more detail!
Good luck!
Regards,
p
pDNA on Wed May 25 15:18:05 2011 said:
pUC vectors and pET vector poses the same ColE1-like origin and are therefore not compatible (although they can be maintained by using selection pressure when both plasmids contain different resistance genes). The f1 origin is only used for the generation of single stranded DNA and is not able to replicate the plasmid in bacteria!!!
The copy number of your vector is a crucial parameter in protein expression. If you use the T7 system for expression of protein even high protein yields can be achieved with low copy numbers ...if you use promoters like lac or tac you can use higher copy numbers. It always depends on your protein and what is your ultimate goal ...high content of soluble protein or insoluble protein in form of inclusion bodies ...or something else.
Maybe we can help you if you will describe your experiment in more detail!
Good luck!
Regards,
p
my aim is to purify two proteins that interact and crystaliise them...one of them i knw from in silico analysis has intermittent large segments of disordered region...and is thus unstable and doesnt properly fold.....I have been unsuccessful with cloning the full length gene. Also even if I am able to clone it doesnt express at all..though the sequence is alright...so I tried cloning fragments..and observed that the N-terminal doesnt express...the middle part expresses but every time I have to induce expression I have to do fresh transformation into the bacteria...no problem with the ending fragment....
So I decide since two proteins interact...the presence of the binding partner may help in proper folding of the other and hence purification....
I cant buy co-expression vectors. I am thinking of two vectors with diff and compatible ori and different antibiotic markers for cloning and thus co-expression in the same bacterial expression strain...I havent done much cloning before and I am confused with the concept vector prep....
I already have the smaller gene construct ( in pET28a) that expresses well...I am struggling with the bigger partner...
maybe you can try to get a compatible expression vector from the japan vector collection ...see this link.
you could e.g. try a pBAD28 plasmid with a p15A origin, inducible by addition of arabinose ...see this link.
...or you could go for any other expression vector that has an origin other than the ColE1ori (in the JPV ColE1=pMB1).
All the best!
Regards,
p
you can try using
rensan on Wed May 25 15:35:55 2011 said:
pDNA on Wed May 25 15:18:05 2011 said:
pUC vectors and pET vector poses the same ColE1-like origin and are therefore not compatible (although they can be maintained by using selection pressure when both plasmids contain different resistance genes). The f1 origin is only used for the generation of single stranded DNA and is not able to replicate the plasmid in bacteria!!!
The copy number of your vector is a crucial parameter in protein expression. If you use the T7 system for expression of protein even high protein yields can be achieved with low copy numbers ...if you use promoters like lac or tac you can use higher copy numbers. It always depends on your protein and what is your ultimate goal ...high content of soluble protein or insoluble protein in form of inclusion bodies ...or something else.
Maybe we can help you if you will describe your experiment in more detail!
Good luck!
Regards,
p
my aim is to purify two proteins that interact and crystaliise them...one of them i knw from in silico analysis has intermittent large segments of disordered region...and is thus unstable and doesnt properly fold.....I have been unsuccessful with cloning the full length gene. Also even if I am able to clone it doesnt express at all..though the sequence is alright...so I tried cloning fragments..and observed that the N-terminal doesnt express...the middle part expresses but every time I have to induce expression I have to do fresh transformation into the bacteria...no problem with the ending fragment....
So I decide since two proteins interact...the presence of the binding partner may help in proper folding of the other and hence purification....
I cant buy co-expression vectors. I am thinking of two vectors with diff and compatible ori and different antibiotic markers for cloning and thus co-expression in the same bacterial expression strain...I havent done much cloning before and I am confused with the concept vector prep....
I already have the smaller gene construct ( in pET28a) that expresses well...I am struggling with the bigger partner...
Are you sure that the selection pressure for two different plasmid sharing the same origin will work.
I want to try pET151 (amphicilin) with pET200 (kanamycin) to coexpress together and the both contain different gene.
thanks
-P
pDNA on Wed May 25 18:09:19 2011 said:
maybe you can try to get a compatible expression vector from the japan vector collection ...see this link.
you could e.g. try a pBAD28 plasmid with a p15A origin, inducible by addition of arabinose ...see this link.
...or you could go for any other expression vector that has an origin other than the ColE1ori (in the JPV ColE1=pMB1).
All the best!
Regards,
p
you can try using
rensan on Wed May 25 15:35:55 2011 said:
pDNA on Wed May 25 15:18:05 2011 said:
pUC vectors and pET vector poses the same ColE1-like origin and are therefore not compatible (although they can be maintained by using selection pressure when both plasmids contain different resistance genes). The f1 origin is only used for the generation of single stranded DNA and is not able to replicate the plasmid in bacteria!!!
The copy number of your vector is a crucial parameter in protein expression. If you use the T7 system for expression of protein even high protein yields can be achieved with low copy numbers ...if you use promoters like lac or tac you can use higher copy numbers. It always depends on your protein and what is your ultimate goal ...high content of soluble protein or insoluble protein in form of inclusion bodies ...or something else.
Maybe we can help you if you will describe your experiment in more detail!
Good luck!
Regards,
p
my aim is to purify two proteins that interact and crystaliise them...one of them i knw from in silico analysis has intermittent large segments of disordered region...and is thus unstable and doesnt properly fold.....I have been unsuccessful with cloning the full length gene. Also even if I am able to clone it doesnt express at all..though the sequence is alright...so I tried cloning fragments..and observed that the N-terminal doesnt express...the middle part expresses but every time I have to induce expression I have to do fresh transformation into the bacteria...no problem with the ending fragment....
So I decide since two proteins interact...the presence of the binding partner may help in proper folding of the other and hence purification....
I cant buy co-expression vectors. I am thinking of two vectors with diff and compatible ori and different antibiotic markers for cloning and thus co-expression in the same bacterial expression strain...I havent done much cloning before and I am confused with the concept vector prep....
I already have the smaller gene construct ( in pET28a) that expresses well...I am struggling with the bigger partner...