Genetics Question?

Question

The specific question was

How would one overexpress an important protein. Include the method used to enable rapid one-step purification.


My answer is

Isolote the gene encoding for the protein and ligate to His-tag. . Then clone modified gene into a LAC promoter in a bacterial artificial chromosome and tranform into E-coli, make sure the BAC has Ampicillin resistance gene. Then select for bacteria having Ampicillin resistance. intruduce lactose to induce LAC promoter. Then remove proteins and apply to affinity medium to which the HIS tag will bind and then wash to remove unwanted proteins. then elute wanted proteins using imidazole.

what do you think of my answer?

Answer 1

You're answer is fine. You could be more specific about the first steps, instead of just isolating the gene and ligating to a his-tag. You'd most likely PCR amplify the gene and ligate that either to a plasmid with a His-tag sequence on it (one step) or in two steps by first ligating it to a TA cloning vector (leaving a few minor details about polymerase and other vectors out because I think it will clutter the answer).

I agree with the first response that you'd want it in a high copy number plasmid designed for protein expression and not a BAC to dramatically increaase the amount of protein produced. Your purification part is fine, but change affinity medium to affinity column (or nickle column, which is what you use for His-tag purification). You might also say how you plan to remove the proteins (sonication and centrifugation) so that your sure that whoever is scoring your answer understands that it's 1 step.

The second response is also valid, you could put a signal sequence on the protein to make the bacteria secret it, but in theory you could possibly end up with folding issues or something of that nature.

These are all just minor details however and your answer is fine, I think.

Answer 2

Pretty good, although I would highly recommend using a high copy number plasmid instead of a BAC. High copy number plasmids will produce much more protein and be easier to manipulate.

Answer 3

If you can add a signal peptide that will cause the protein to be excreted across the cell membrane then all you need to do is spin down the cells. leaving you with a dilute solution of the protein