I want to ligate two protein coding genes (by any means ie., using linkers or some other way) without affecting their native structure and function when they are expressed. Any suggestions?
I think you need a cloning strategy in the first place. You might insert both of your genes in a cloning vector. You would also need to have a space between the two genes, and the best way of doing this is to have a non-coding region in between the two, including restriction sites flanking the ends of your genes. This is particularly important if you want to further manipulate your genes for other uses. I attach an explanatory image of a vector containing a GFP gene, and our gene of interest as well as 3 enhancer regions being inserted in the vector. This way you can link/ligate and co-express two or more genes in a single vector. I hope this would help. Regards,
I would use flexible linker (gssgss.. etc) between the two proteins.
Be ware that "A-linker-B" and "B-linker-A" fusions might have different properties,
I'd try both. If you want to purify the fusion protein, place orthogonal affinity
tags at C and N terms, this way complete construct may be purified with ease.
Question is, you want to express two proteins as a fusion protein or you want to express two proteins under the control of a single promoter (bi-cistronic expression). If you mean expression as fusion protein then Shad and Misha have good suggestions. If it is about bi-cistronic expression then IRES and P2A is something you would like to consider.
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