How do I change insoluble recombinant proteins to soluble proteins? My recombinant protein is Aldose dehydrogenase from bacterial with around 35.0 kDa size. Now, My expression vector and strain are pET22b and Bl21(DE3), respectively. And, I have already tried to use IPTG (0.2, 0.4, 0.5 mM) in LB media to induce it with OD600=0.8 at 18, 20, and 25°C for 16h, respectively. In addition, I also tried to used auto-induction method (lactose, T7) by ZY media at 25°C for 16h. all of the previous results showed that my purpose protein is insoluble.
Now, my supervisor suggested me to try to use other expression strains to express again. So, I have tried to use Omp8, C43(DE3), M15, Origami and Rosetta(DE3) to do again but now I am waiting for the expression.
By the way, for lysis buffer, I used 20mM Tris-HCl with 300mM NaCl and 10mM imidazole, pH 6.5 (my protein pI is around 5.5).
I will upload my SDS-PAGE gel later.
Thus, How Can I resolve this problem by any other potential method or any suggestions? thank you every much!
For papers and reviews about recombinant protein production, you may check the papers by A. de Marco, e.g. A. de Marco Methods Mol Biol. 2017;1586:197-210. doi: 10.1007/978-1-4939-6887-9_12. Your problem is indeed tricky and frustrating, but by no means infrequent. Some proteins never consent to be produced in soluble form in E. coli. If the gene you trying to express comes from a eukaryotic organism, it may be worth switching to a eukaryotic expression system, such as insect cells, Leishmania tarentolae, or even mammalian cells. Sometimes, switching to a homologous gene from another, related organism can also help, if it si compatible with the questions you plan to ask with the protein
Firstly, changing of expression strain sometimes helps in expression of protein in soluble fraction. Considering your protein is overexpressed, not getting the protein in soluble fraction means that it's forming inclusion bodies. In such a case, the cell pellet will contain the desired protein (which can be observed in an SDS-PAGE). If so, you can try to purify the protein under denaturing conditions (urea/Gdncl) and then refold the protein slowly by repeated dialysis change.
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