I am trying to purify a recombinant protein for its functional study and I want to do some biochemical assays. Due to hydrophobic nature of the target protein most of its fraction forming inclusion bodies. I have tried various experimental conditions to increase the protein concentration in soluble fraction like induction at different temperatures and IPTG concentrations, tried with different tags ,e.g., His, GST, MBP. With MBP tag the protein concentration in soluble fraction increased to very little extent. But this increased concentration with MBP tag is not sufficient to subject the protein to second column purification like Gel filteration or ion exchange. Solubilization and refolding of protein from inclusion bodies is a tricky process and the protein may not be functionally active. So I am thinking of in vitro transcription translation system. Is it possible to purify the target protein after its synthesis by in vitro translation system so that it can be used in biochemical assays. Thanks in advance.
It is certainly possible to purify a protein made by in vitro transcription/translation. There are commercial products for producing usefully large amounts of protein by this method. You could include detergents or chaperones during the protein translation, perhaps, to help the protein to stay in solution or fold, as suggested here:
http://149.250.246.19/wcsstore/RASCatalogAssetStore/Article /BIOCHEMICA_3_01_p27-28.pdf
Dear Ajay,
As Adam said, there are commercially available kits for producing protein in such a way. My (admittedly limited) understanding of these systems is that the level of purification required is low; as in theory you should only be producing the protein of interest.
However, it is more complicated to produce membrane proteins or anything subjected to numerous post-translational modifications in this way. Additionally, I would suggest cell-free expression as an option if you were having problems with toxicity. Your protein sounds as if it's expressing ok, just not in the soluble fraction - do you expect a cell-free system to change that? Have you explored other recombinant expression systems? There are strains of E.coli that may be better suited for your protein, or you could explore insect/mammalian expression.
Best wishes,
James
Recombinant HIV Reverse Transcriptase had posed similar problems; it was solved by putting in point mutations that would allow retention of RT function but increase its solubility. Bob Craigie's group at NIH used this approach and crystallized the protein for its first crystal structure. David Davies' group did the X-Ray Crystallography. It is old work and I think similar approaches have since been used for difficult proteins.
Hello Ajay
Did you also attempt the expression using a different (weaker) promoter. In my experience (which is definitely limited) it should be possible to tweak the system using a weaker promoter which would allow lower expression levels, but will probably give a better yield of soluble protein. Also, have you considered co-expressing chaperons with your protein as a possible solution?
I hope you find the attached article useful.
VBW
Shahan
- Badges
- Science method
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Methods
- Protein Purification Techniques