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Protein folding problem

protein folding e.coli vegf western blot

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#1 Randy Smith

Randy Smith

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Posted 11 November 2012 - 06:43 PM

Hi all,

I have a question regarding protein folding. I have tried many different ways and finally found an E.coli strain (from NEB Shuffle) that has a DSBc shuffling protein.
I have been trying to get e.coli derived VEGF-165. It has 7 disulfides, two of which form a cystein knot motif forming a homodimer of two 19.2kDa monomers.
I have tried expressing with low IPTG, adding sucrose (0.4M), adding reduced glutathione (10mM) (this "recharges" the DSBc...a few publication reference this supplement) and expressing at 15C overnight. I use a broth that seems to be a mix of TB and LB (recommended by NEB).
I extract via lysozyme and sonication and I run the lysate on a Histrap column (have tried with Ni and Cobalt).
The SDS Page and WB show a monomer at the expected size of 25kDa (I have some modifications to the N terminal, which include a 6xHIS, thrombin cleavage, a peptide sequence NQEQVSP, and 2xGGGS linker).
However the SDS and WB also show a "dimer" at 35kDa...which is much lower than expected. I have run these against commercially derived VEGF165 (20kDa monomer, 40kDa dimer via SDS/WB).

I have no idea why this is happening, bc the commercially derived bands are at the expected size.
Is it possible that the e.coli is forming dimers with non-vegf proteins? When I run under reducing conditions the WB does not show up (expected, my Ab does not recognize reduced via DTT). But the SDS barely changes in location of the bands.

Here is another twist. The protein is only active in the dimer form, yet when I do a HUVEC proliferation assay I get the same (slightly lower) activity as compared to the commercial VEGF. For instance my control with no VEGF when seeded with 1.5K cells increases to 1.7K cells over 3 days with 1%serum. My engineered VEGF (at highest concentration tested vegf) increases to 2.7K and the commercial vegf increases the cells to 3K cells.

I am going to try to fully reduce via DTT the bacterial lysates, run over histrap (all in presence of DTT) and then during the elution step elute with no DTT into a solution containing the 1:5 ratio of reduced and oxidized glutathione, hopefully this will refold with the correct disulfides (I hope).
Do you think this will work?

BTW I have tried producing as inclusion bodies and refolding, and despite many publications claiming refolding, I have never been able to refold from IB's.

Thanks!!





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