I want to express my protein of interest which forms disulfide bonds into inclusion bodies in E. coli and then refold it. This works completely fine for related proteins from this family but for one of these proteins I had no protein expressed in inclusion bodies although this should happen (reproducible). The gene is codon optimized for E. coli and I use a T7 promoter for expression at 37°C. Has anyone an idea why this single protein is not forming inclusion bodies although it should?
You mean no protein expressed into inclusion bodies, or no protein at all (i.e. no soluble expression either)?
Maybe your protein is not expessed at all or it is degraded after expression. Check your construct and add a cocktail of proteases inhibitors
I have not checked for soluble expression as this should not happen due to the necessary disulfide bonds for correct folding. The construct is OK, I checked the sequence of the plasmid at the end of the cultivation. Could a degradation happen cotranslationally and if so, how could you stop this?
Denatured proteins do not always aggregate into insoluble inclusion bodies. Checking the soluble fraction is a simple enough experiment and might save you time spent looking for alternative explanations.
If there is nothing there then your target protein simply isn't being expressed at high levels. Most of the time (if transcriptional induction is OK) this is caused by inefficient translational initiation. Analyzing your construction with something like the RBS Calculator at https://www.denovodna.com/software/ might give you a clue as to whether this is the case.
Thank you all for your answers. I will check for potential soluble expression first and in the next expression I will use PMSF additionally. To overcome potential problems with mRNA stability etc. I will use BL21(DE3)Star strains as well as the 5' UTR of the ompA gene (construct is already cloned). I will keep you updated :)
Hi Jonas
the insoluble fraction was isolated, denatured and refolded. See below.
The pellets were dissolved in 6 M guanidine and 50 mM Tris–Cl, pH 8. The recombinant protein was purified by affinity chromatography on Ni column (HiTrap, GE) preequilibrated with 6 M urea, 50 mM Tris–Cl (pH 8.0), 300 mM NaCl, and 5 mM imidazole. The fractions corresponding to the target protein were pooled and dialyzed overnight at 4 °C against the following buffer [6 M urea, 50 mM Tris–Cl (pH 8), and 300 mM NaCl] to remove imidazole. Afterwards, the sample (8 mL at 0.6 mgmL−1)
was reduced with 10 mM of β-mercaptoethanol for 1 h at 4 °C and diluted drop by drop into 500 mL of refolding buffer at pH 9.0 (50 mM 3-cyclo-hexylamino-ethylsulfonic
acid (CHES); 10 mM β-mercaptoethanol and 10 % glycerol) previously chilled.
Update: ompA gene and BL21(DE3)star strains did not solve the problem so mRNA stability was not the main problem. The gene seems to be toxic so we could only grow colonies that express at low levels. Problem could be solved by using E. coli Lemo21 strain adding chloramphenicol to the preculture. In addition, we mutated the construct and clones with two additional N-terminal amino acids worked better.
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