Hi Dawid...purifying an active eukaryotic protein from bacteria is generally difficult and in ur case it is not even a soluble protein. But u cn try for sure.

use E coli BL21 cells...overexpress ur protein and induce at low temperature (this is a "must do" step) overnite (12-16 hrs, 1 mM IPTG, temp. 16C). then keep the pH of ur solutions basic (Tris 8-8.5) throught. then usual procedure ---harvest, lyse, remove the soluble fraction (u can keep this jus to check in case ur protein comes in soluble fraction also...yes this is possible when u induce at low temp). After removing the supernatant, take the debris containing inclusion bodies..redissovle this fraction in buffer containing- Tris pH 8.5, NaCl, protease inhibitors, beta-ME, N-lauryl sarkosine (1-2%), and triton X-100 (2-4%). keep ur sample in this buffer for 1 hr on ice. now again remove the debris and take the soluble fraction to bind to ur resin (i suppose u r going to use Ni-NTA). now go ahead with ur procedure---binding, washing, elution with 200mM imidazole. We generally do not try to refold it. all the best

Hi Dawid,

Andaleeb is correct, over expressing proteins in E. coli can make some trouble. The fact you have inclusion bodies is a mixed blessing. You can very quickly and easily purify your target protein by simply washing away the soluble material.

I did this working with human IL-7. To refold material from inclusion bodies can be difficult. I denatured the inclusion bodies containing my target IL-7 protein in 6 M guanidine. Then I either dialyzes the denaturent out or refolded by dilution. Basically the polypeptide "knows" how to fold up; if you can give the polypeptides enough room they will fold and some percentage of material will be biologically active.

The dialyzed or diluted material could now be used for binding to your resin for further purification or assayed for TGF-alpha activity.

Do you need the tags? Maybe eliminating one or both may help with solubility issues. But than you may have purification troubles.

Another approach is to use an expression vector that secrets target gene expression products out and into the media. In these systems the secreted material will most likely be correctly folded and biologically active and relatively easily purified.

Good luck and best regards,

Larry

Hi Dawid,

Aside from refolding enriched inclusion bodies in solution, you cold also try to refold your protein directly on column. Since you express your protein with a His and S-tag, you would bind your recombinant protein to the appropriate matrix and denature it as you would do in solution. Then you carry out the refolding with protein bound to the column and afterwards elute it or if your vector permits cleave the protein of the tag (thrombin site or equivalent).

Good luck,

Philipp

Thank you all for your answers.

Generaly I was trying to obtain it using many conditions( lower temperature, different IPTG concentrations, differnt lysis buffers, different strains) and it was not soluble. In literature I didn't found any case of expression of soluble TGF-alfa with or without fusion partner. So generally I lost hope of obtaining it soluble.

I think that I will have to refold it anyway. I am simply afraid that it won't fold becase of long fragment with tags.

Also, I will have to remove the tag with enterokinase. Do you know if it may be possible before refolding? Because I am quite sure that I will refold it to native state without N terminas tags (my labmate did it with very similar protein).

Hi Dawid,

It is usually better to remove the tag before refolding, even though it can work with tag in some cases. You should be able to do it after denaturing, but the efficiency might be quite poor. If you are looking at purifying inclusion bodies anyways, it is likely easier to express the protein without any tag to avoid that problem.

Cheers,

Philipp

I think one of the problem may be the three disulfide bond in TGFalpha. Did you consider secretion/periplasmic production? Protein produced as inclusion bodies usually does not contain the disulfide bridges, and getting three disulfide bridges correctly formed during refolding is difficult. You really have to play around with redox shuttles during refolding. There are bacterial strains available that have been engineered for improved disulfide formation in their cytoplasm:

Origami™ 2(DE3)pLysS Competent Cells - EMD4Biosciences | EMD ...

http://www.emdmillipore.com/life-science-research/origami-2de3plyss-competent-cells/EMD_BIO-71346/p_FUKb.s1Oh6IAAAEjTBl9.zLX

However, with antibody scFv we generally found it easier to produce them by periplasmic production than either in origami strains or by refolding.

This paper describes the production of EGF in bacteria:

ScienceDirect.com - Enzyme and Microbial Technology - Cultivation ...

http://www.sciencedirect.com/science/article/pii/S0141022906003152

by H Zhang - 2007

Cultivation of recombinant Escherichia coli for secretory production of human epidermal growth factor under control of PL promoter. Haiyi Zhanga,; Zhimin Lia, ...

As EGF is structurally very similar to TGF alpha, you might search for papers on microbial production of EGF for additional approaches to the problem.

Thank you very much Annemarie!

This publication is very helpfull. I was looking for papers about expression of EGF in E.coli but I found one and it didn't give me all the answers I was looking for.

We were using RG E.coli and it didn't help. I am thinking about periplasmic expression all the time, but I simply wanted to ask if it's possible to use this construct with His- and S- tag simply because it is ready and expression is present( TGF alone didn't give expression).

If it's about refolding of naked TGF it's possible and all the papers I found telling about TGF expression included its refolding .

I think I will try to refold it anyway, and if it won't work I will think about preparing a new construct.

The sequence of the first few bases after the start codon can have a strong and difficult to predict effect on translation initiation. mRNA structure may be a reason for this. As a result, more or less everybody uses the same few tags that were found to work well.

Of course in secretion, the translation initiation is governed by the signal sequence, which is cleaved off during export.

This is a useful database about refolding procedures:

http://refold.med.monash.edu.au/

The REFOLD database: a tool for the optimization of protein expression and refolding.

Chow MK, Amin AA, Fulton KF, Fernando T, Kamau L, Batty C, Louca M, Ho S, Whisstock JC, Bottomley SP, Buckle AM.

Nucleic Acids Res. 2006 Jan 1;34(Database issue):D207-12.

“Protein Expression and Refolding – a practical guide to getting the most out of inclusion bodies”, Cabrita and Bottomley, 2004, Biotechnology Annual Review, 10:31-50.