Using pB2GW7,0 - (Jan/16/2017 )
Hi all, I need help with the following.
I want to get a mutated protein expressed in plants.
If I have the relevant nucleotide sequence, how can I get a new DNA sequence TO A TUBE, without commercially synthesizing it?
Are there any vectors which can be used both for cloning and
overexpression of plant proteins?
I came across pB2GW7 vector. The literature says that it does not have a tag. Can we add the tag to the primer or is there any other way to purify the protein produced if its produced with out a tag like 6 His.
You can clone the sequence from a cDNA source, but other than that you would need to have it synthesized in some fashion. Synthesis is getting cheaper, its down to about $1 per bp now.
There are cloning and expression vectors in the animal and bacterial worlds, so I would guess that the same applies for plants. However, it is often easier to clone in a small vector such as one of the pUC series, then transfer this to an expression vector.
You can add the tag to the primer. The tag is not involved in the annealing temperature calculation. Make sure that you add the tag at the 5' end of the primer that you want it on.
There are tag-free purification systems, these often rely on antibody based affinity purification, but you can also do such things as size exclusion, ion exchange, and chromatofocussing. GE lifesciences has some good (free) resources on this.