Asp was substituted for Glu. SDS-PAGE revealed that the yield of the mutant protein was lower than that of the normal protein and the yield of inclusion bodies in the mutant protein was higher than that of the normal protein. Bioactivity assay revealed that the mutant proteins were inactivated. I wonder if the amino acid substitution can induce a decrease in protein expression
Single AA substitutions cause a number of profound disease states, including sickle -cell anemia, so yes they can also have profound impact on expression level, folding and cellular distribution. I'm also aware of rare codon induced translational pausing which can do similar things. Lower expression coupled with higher percentage in inclusion bodies suggests the folding perturbations induced are quite efficient at getting your mutant shuttled to the "quality control" machinery within your expression host. While its a quite similar AA it has a big impact, suggesting its probably highly conserved and for good reason.
Hi TienLi,
Therory:
there will be the impact on expression and protein stability depending the amino acid position,
poor or high expression is mainly because of the amino acid preference by the host, ribosomes for faster synthesis and translation.
m-RNA open structure in the first 30nt and relatively closed structure at c-3' influences the expression levels.
Practical:
have you observed any reduced cell growth?, is there any leaky expression?
if yes, pls reduce IPTG conc to 0.1mM and check if the results are changing
lastly, as we commonly use the same std media as LB , we observe these changes, and media optimization shall improve protein expression from current state
Dear Tien Le
Sobstitution of Asp with GLu is quite conservative sobstitution and in the most of cases it does not affect conformation and stability of a protein but each protein has its own properties and it is possible that in some cases it may alter the protein stability or ability of the protein to bind a cofactor and therefore activity and stability. Expression yield is ofter direclty correlated with stability, since a poorly stable protein is degraded and result in lower expression.
If a PDB file is avaialble for you protein you can try to predict the effect of mutations using some software as Popmusic
https://soft.dezyme.com/
or you can try also to verify it by comparing the thermal stability of the purified proteins (WT vs mutant) with DSF which is a simple and fast tecnique that you can easly run if you have in your lab an RT-PCR instrument.
https://www.blogger.com/video.g?token=AD6v5dzr9KqZslTx9NeEjXx1XyPti8dU15eqYoE9TT8OEDaZQcNINuDKwzCAzVXkFWZA0btipKTr81doABh-VjpprwCcRukVcLhAXE_T5BzPxYbeQQA7VMFmc4DdpjS4XbOfxkwAhIWu
best regards
Manuele
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