Randy Smith

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!!

Hi all,
New to posting in this forum but I have been actively reading for over a year. Now I find myself in need of an answer for a question.

I am expressing VEGF protein in E.coli (special from NEB that forms disulfides). VEGF is a homodimer and I have modified in via cloning too include a 6x HIS tag, a Cystein "tag" (one Cys amino acid) and 2x GGGS linker at the N terminal (C-term is active end).

I am getting good expression but odd SDS-Page and WB results.
My dimer should be at 50kDa (calculated) and unmodified VEGF runs at about 40kDa dimer.
My dimer is showing up at 31kDa and a monomer just under 20kDa.
This is very obvious in a western blot and not so obvious in SDS Page (dealing with purification).

Is this common?

I am not using reducing conditions for the SDS Page. Boiling for 5 mins at 95 in sample buffer.
Ladders are biorad broad range (not prestained) and I am using biotinylated ladder for the WB.

The unmodified VEGF monomer is 19kDa, with the dimer between 38 and 40kDa, but I have added the 6xHIS, thrombin cleavage site, a cys residue and 2x GGGS linker prior to the VEGF.

Checked sequencing and it is correct and has no frameshift mutations.
Antibody is monoclonal from R&D.
Does not detect reduced dimers in presence of DTT