Biochemistry help

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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Biochemistry help

Post by rekha1 » Sun Apr 04, 2010 8:37 pm

I am student working on Biotech project. I am trying to make an artifcial metalloprotease with CoIII as catalytic center aiming at cleaving V-L bond in a peptide fragment in body. Can someone help me with engineering this enzyme as I have very little knowledge in biochemistry

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Post by david23 » Mon Apr 05, 2010 4:32 am

wow you certainly know how to use the right words. Are you sure you dont know much about biochem

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Re: Biochemistry help

Post by domwood » Mon Apr 05, 2010 7:07 pm

I don't know anything about your specific enzyme, and I may be mis-understanding your project, but I guess from a Molecular Biology standpoint the route I would follow would be:

1) Select the sequences you want included to make your enzyme, the metalloprotease sequence and ColIII sequence.
2) Design primers to amplify the sequence with PCR, I imagine the minimum you'd need is 3 sets one for metalloprotease 5' of where you want the catalytic center, 3' sequence, and the ColIII sequence.
3) Design the primers with restriction sites in them (make sure they're unique)
4) The sequentially cut&paste the sequences into a vector.
- If you actually want some enzyme to be expressed that is a big task.
5) If you want it to be expressed in the 'body' then you'll need some sort of viral vector or liposome (Not really my area). GM would be an easier solution but I imagine your talking about Humans, in which case big No No.
6) I would think in might be easier in the long run to express the protein via a yeast expression system. Then inject it into human cells. Either way certainly not a walk in the park, could take years even for a trained molecular biologist. I doubt you would be able to do it with just some forum posts, you need to actually be trained.

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