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Clauelena13

Member Since 17 Sep 2012
Offline Last Active Sep 18 2012 11:18 AM

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Claudia Elena

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protolder
20 Sep 2012 - 13:22

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Peptide recuperation after FactorXa cleavage using GST fusion

18 September 2012 - 11:18 AM

Iam tryng to express a peptide of 6kDa using a GST fusion in E.coli. After cleavage using FactorXa protease, peptide precipitates with the GST...I have tried using reducing buffers with b-mercapto after cleavage (not before because factorxa is sensible to reducing agents), as the peptide have a Cys residue that may be forming di-sulfur bridges...It clarifies the precipitate, but according to HPLC results it seems that the peptide continues in an aggregated form. HAve you any experience in order to redisolve the aggregated proteins? I think to try with some caotropic agents such us urea...but I have no much experience in this field.
Thanks !
Clau

Peptide concentration methods

18 September 2012 - 10:42 AM

I have been trying to produce a 6kDa peptide through two different strategies. One of them is using Pichia pastori secretory production. In this case, the problem is that I cannot detect the 6kDa peptide in the media...it could be that the pichia clones obtained are not good in terms of production, but to considere this I first have to discard that the peptide is been produced in levels only seen when concentrating the media...have you any ideas about peptide concentration methods using in the broth media? I believe that ultrafiltration is not suitable because of large volumes I need to use...What about precipitation methods? HAve you any experience in this point? Iam not sure if TCA precipitation works well with peptides.
Thanks in advanced,
Clau