I am designing primers to get a soluble construct of a protein by removing the signal sequence from N-terminal region and membrance anchor region from C-terminal of the protein. I am using pET28a expression vector and I want His tag at N-terminal region for purification. The vector itself has spacer region and initiation codon upstream of His tag sequence. Do I need to add ATG sequence in the forward primer of my gene sequence as well? Will the presence of extra ATG sequence affect the expresion of gene with His-tag?
Can anybody clarify. Thanks
Hi there,
You don't need to add an ATG codon in your primer. The only things to be careful about are :
respecting the reading frame between vector and insert
insert an in frame stop codon in your reverse primer
The presence of a second ATG shouldn't affect protein expression level. However position of the tag(Nter or Cter) may affect it...
No, you don't need to add an additional codon in your primer. In addition, you should care that your primer or the distance between the position of your primer and the start codon of your gene has no stop codon.
When designing a forward primer for protein expression in E. coli BL21 cells, whether you need to include an initiation codon (ATG for methionine) in your primer depends on the design of your expression vector and the specific requirements of your construct.
In summary, the need to include an initiation codon in your forward primer largely depends on the design of your expression vector and how it aligns with your gene of interest. Always check the vector's sequence and the cloning strategy thoroughly before designing your primers.
l This protocol list might provide further insights to address this issue.
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