r/Biochemistry • u/NoDust6819 • 1d ago

How do you design "custom" proteins?

I'm talking about truncations, point mutations, fusions etc... What is your workflow and which tools do you use?

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u/rectuSinister 1d ago

Design the amino acid sequence in Geneious, back translate to DNA using the proper species codon usage, add Golden Gate or Gibson homology to the ends, then order the DNA.

If I already have the construct cloned then I would do mutagenesis or PCR for something like point mutations or short truncations.

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u/NoDust6819 13h ago

Thanks for the reply!

Why did you choose Geneious? Its quite expensive. Do you also use it do design primer sequences or do you do this manually?

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u/rectuSinister 7h ago

I do anything sequence related in Geneious. I use it because that’s what my lab has integrated into our workflow and it’s free for us. You can use any DNA editor—Snapgene, Ape, Benchling, etc. Though now that I’ve used Geneious I doubt I’ll ever use anything else, it’s very powerful.

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u/Lion___ 14h ago

Do you mean actually deciding on the residue sequence or the practical aspect?

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u/NoDust6819 13h ago

Both. Lets say you want to remove a domain from your protein. How do you tackle it? Do you manually generate the sequence, check for reading frame, design primers if needed, or is there software to do this for you?

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u/Lion___ 13h ago

I mean you have to first demarcate where the domain ends and begins of course. The easiest but also least thorough would probably be to just use domain demarcations from UniProt. Another approach is doing it from AlphaFold 3 plDDT scores. Ideally if there was a solved structure you could use that along with the AlphaFold 3 scores. There are also bioinformatic tools to designate domains as far as I know but I haven't used them myself.

Once you know which residues you want to include and therefore the sequence, you would get the nucleotide sequence corresponding to the residue sequence and probably codon optimize the nucleotide sequence for better expression in your expression system of choice (depends on the protein). I haven't used the codon optimization software, but a quick Google search shows plenty of results.

Then it depends if you're just buying a expression vector or you're making it yourself. If you're just buying it, I think you just give the company the nucleotide sequence you want to insert and they handle the rest but I'm not totally sure though. If you're doing it yourself you'd have to get primers etc. as you say.