There are a number of ways that you can do this and even kits available to help you with the cloning. You can also add polyA tails by incubating your PCR product with Taq polymerase at 72C for 30 minutes with dATP only and ligate into a cloning vector suitable for polyA cloning or ligate the PCR product into a blunt cloning vector, then digest with appropriate restriction enzymes and ligate into a pET vector but I find this to unnecessarily add extra time. I tend to skip this step and clone directly into the expression vector using restriction enzyme sites as it give me directional cloning, in a cloning single step, ready for protein expression.

I usually use Phusion polymerase (for low error rate) and design the primers so that they contain a restriction site each, followed (or preceded) by another 10 base pairs of random sequence to allow enough length for the restriction enzymes to cut. You should choose the restriction sites from the multiple cloning sites of the pET vector. One strategy is to find the start codon in the vector and choose the restriction site that includes the start codon e.g. I used pET22b and in that vector you could choose Nde I then in your primers start the clyA sequence immediately after the Nde I site leaving the ATG out as it is already contained within the Nde I site. For the 5’ end the restriction site immediately before the 6His tag can be chosen - XhoI in pET28b. Also you should remove the stop codon from the clyA gene itself to allow translation into the pET vectors sequence containing the 6His tag at the end. This should produce your protein of interest with as few as possible amino acids (two only) at the C terminus followed by the 6His tag. In terms of other details like the RBS, etc they are already contained within the pET vector and as long as you keep the existing position for the start codon they should be fine.

After PCR amplification I usually digest both the pET and the PCR product with the chosen restriction enzymes and then ligate into the pET vector directly using a general purpose cloning/expression strain (usually E. coli XL1 blue, commercially available chemically competent cells). Sometimes I incubate the vector with phosphatase to prevent re-ligation on itself but that should not be a high frequency event if you use different restriction enzyme sites.

I hope this helps.

Dear Cosmin

Hi

I hav the same problem, l want to clone interest gene in NdeI of pET 21a , I dont sure abuot ATG of Nde1

As start codon but some friens sayid it acts as start codon and inresult I must delet the our interest gene statrt codon? Is it right? And one more thing If we use Xho1 site in the end we will have Glu and

Lu as 2 addition aa in protein , in your opinion do these aa effect conformation and function of interest

Protein?

Thanks

Dear Abbas,

Apologies for the delay in replying. If you would like to keep the protein as close as possible to its original sequence I would then take out the ATG in the protein as if you leave it, the ATG in the NdeI site will also be translated and the protein will start with two methionines rather than one. You can take it out by omitting it from the primers that you design for cloning your gene of interest.

You are right about having the two additional amino acids at the end of your protein but I do not think there is much you can do about that given that that is the design of the expression plasmid. It is difficult to predict how much they will affect conformation and function but if that is a worry, then the 6xHis tag in itself may affect conformation and function, possibly even more so. Given that they are only 8 amino acids it is likely that you protein of interest will be unaffected so I would go ahead cloning it and testing it for function after it is purified. There are certain online programmes that you could use to predict the structure with and without the additional amino acids at the end of the protein - I am not too familiar with protein structure predictions but I used Phyre2 and it is relatively easy to use.