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I am currently working on this bacteriophage P2 protein called gpX, but the expression pattern is wierd. This phage infects and kills E. coli efficiently, so codon bias should not be a problem here. The expression conditions I have used are as below:
1. TB media at 37C for 4 hr --> Good expression BUT insoluble protein (inclusion bodies)
2. TB media at 16C O/N --> Good expression AND soluble protein YAY!!!
3. LB media at 16C O/N --> Fair expression (~1/4 O/N expression of TB) AND some soluble protein
4. M9 media at 16C O/N* --> ~1/2 O/N expression of LB AND some soluble protein
*The supplements in my M9 media includes MgSO4, CaCl2, Biotin, Thiamine, FeCl3. The C source is glucose and N source is NH4Cl.
I believe that the increase in overall expression in TB is mainly due to the higher cell density, and the reason I wanted to increase the protein yield in M9 is that I would like to solve the structure of this protein by solution NMR (since its only 67 residues).
Through bioinformatic analysis, I have found this protein to contain a putative peptidoglycan binding domain (the lysM domain). Moreover, CD temperature melt studies on 20 and 30 uM of the protein (purified using native prep) show that it is probably not very folded, but this is not conclusive and I can do a 1H-NMR experiment to completely determine this.
But if the protein is fairly unfolded in the cell, it could be degraded during expression. This would have to be complete degradation as I see no major degradation product in my protein prep. And possibly, it gains it structural integrity from binding to peptidoglycan (I will do a peptiodglycan binding assay next week to see if it is peptidoglycan-binding). I have checked the literature on lysM domains and have yet to come across such an issue in the publications. Moreover, if this is the case, it would be fairly costly for structural determination since free peptidoglycan in E. coli is low compared to recombinant proteins. Maybe some one from the forum can give me further insights?
Edited by laGGer, 25 March 2009 - 07:40 PM.
