Hello,
I am trying to express the VHH that is discovered in our lab. The isoelectric point of the VHH is 7.75 and I tried using pH of 8.00 and 6.8. The gene of interest with His tag is inserted in the pET22b(+) vector and is transformed into BL21(DE3). I use IPTG to induce the protein. The gel ran for pre and post-induction samples shows no difference between pre and post-induction bands. Assuming the VHH expression is very low, I purified the protein with IMAC and SEC. The bands below in the image are the bands from concentrated protein after SEC purification. I also tried protein purification with ammonium sulfate precipitation. It did not help that much and the protein expression is significantly low. My question is why do I see multiple bands with high molecular weight even after SEC purification? And also, why the protein expression is very low? I appreciate all the answers and feedback.
Thank you.
Sashi
Dear Sashi Kandel
ddi you tried to run the purified VHH in non reducing SDS-page?
Are you sure that is it monomeric and in SEC run at 13KDa and not aggregate and run at higher MW (eg 26Kda or more) which may explain why the SEC is not able to separate it.
i wrote this since to my knowledge the VH contains a conserved disulfide bond common to Ig folds found between residues Cys22 and Cys92.
if i understand well, are you using the pet22b, so are you expressing the VHH in the periplasm? How do you extract it? osmotick shock?
Because, since E.coli differently to the mammalian is not very able to form S-S bonds, is it important to understand if youw VHH is produced with the intact S-S bond or not.
best
Manuele
Hello Sashi !
About low yield of protein, you could try various IPTG concentrations and other conditions (initial OD, culture times) to fine-tune expression. You did not post/suggest which protocol you are using and what you did try so far.
And yes, if the next marker is 25-26 kDa, it looks like you have spontaneous dimerisation of that protein, even after SEC.
All the best !
Paul
What are the "I tried using pH of 8.00 and 6.8"?
For ammonium sulphate precipitation you need high total protein concentration, otherwise the proteins will not precipitate.
If Manuelle is right and you need disulfide bond, E. coli is not very good expression host. Why not try some eukaryotic host like Saccharomyces or Pichia?
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