Expression of peptide containing alternating d- and l-amino acids - (Jul/25/2006 )

Hello,

does anybody know something about an publication, written about the recombinant expression of peptides containing alternating d- and l-amino acids? - Or does somybody have some experience in that topic???

Thanks

Rocher

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I would take synthesis route. It is much more difficult than you think it is.

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Thats another problem- the peptides from the synthesizer are not long enough.

I found, that bacillus brevis is producing some antibiotic peptides that contain d- and l- amino acids. You think there is no system availabe, based on that bug?

Rocher

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Its made by a very complex protein system with many subunits, not the usual RNA/ribosome system. The term of expression would not apply here.

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What do you mean when you say the peptides from the synthesiser aren't long enough?
As for B. brevis, and for most (??all) bacterial systems, maybe the peptides are synthesised as all L-amino acids, and specific residues are converted to the D-form by isomerases, rather than being made of both L- and D-forms.

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[/quote]
What do you mean when you say the peptides from the synthesiser aren't long enough?
As for B. brevis, and for most (??all) bacterial systems, maybe the peptides are synthesised as all L-amino acids, and specific residues are converted to the D-form by isomerases, rather than being made of both L- and D-forms.
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Thanks for your answer. We planed to produce peptide containing up to 60 amino acids and they should contain d- and l- forms. This would be quite long for an synthesiser. I didn`t know, that B. brevis turning the l- into d-form by using isomerases. I thought, I could add an high amount of d-aa to the media and B. brevis will use them.

Rocher

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Here is a reference for everybody who is still interested in that topic:

1: Dedkova LM, Fahmi NE, Golovine SY, Hecht SM.
Enhanced D-amino acid incorporation into protein by modified ribosomes.
J Am Chem Soc. 2003 Jun 4;125(22):6616-7. No abstract available.
PMID: 12769555 [PubMed - indexed for MEDLINE]


It^s not made for simple high level expression but it seems to work.

Rocher

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In order to do what you proposed to do I think you need whole set of alternative codon/d-aa-tRNA to do so. by the time you achieve this, you have made your peptide by synthesizer already.

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I've just looked at the abstract. The process appears to be somewhat arbitrary (but a very clever idea). How will you know which residues have D-amino acids substitutions? What would you like to do with these peptides? How would you purify them to homology to work out what they do?

I went back into Pubmed and found an old paper about B. brevis being able to naturally incorporate D-form aa's (J Biol Chem. 1955 Jan;212(1):461-7). Have you considered trying to use B. brevis as is to express your peptides? I have no idea about workign with brevis, nor the paperwork you'd need to go through to do so, but it sounds like a really interesting set of expts.

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Agreed, I dont see how that method (J ACS paper) could control D-isomer incorporation in a specified position, alternating as required.

Are there any internal cys residues? How about two synthetic ~30-mer peptide join together by "native chemical ligation" method?

Anyhow, this is going to be a very expensive project.

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