I am currently studying how to introduce mutations to improve the expression level of proteins.
My lab has a system that can model the structure of proteins through alphafold, which models mutants and calculates the difference in protein fold energy between wild-type and mutants through foldX.
However, when protein mutants calculated to be more energy stable actually expressed, they did not lead to improved productivity. I know that this is a very complex problem with many issues to consider, such as codon bias, mRNA secondary structure, aggregation propensity, etc. Recently, there are many excellent protein modeling tools, including alpha-folds, and specifically I want to know the correlation between the calculated fold energy and the expression level.
Can you tell me if there is any reference that deals with that issue?
Thank you for your interest
The problem is that in real life, there is not just an equilibrium between the unfolded and the folded state whose equilibrium constant is described by the change of Gibbs free energy (delta delta G) of folding.
Folding to the native structure in a real world competes with aggregation of unfolded or misfolded states of the protein, and this aggregation is frequently irreversible. As part of the Plückthun group, I have studied this for antibody single chain fragments, which, upon periplasmic expression in E.coli either folded to the correct structure or aggregated to form periplasmic inclusion bodies, ranging from sequences that landed to 100% in aggregates to others that folded to 100% in the native state, with everything in between.
For example, see: Nieba, L., Honegger, A., Krebber, C. and Plückthun, A. (1997) Disrupting the hydrophobic patches at the antibody variable/constant domain interface: Improved in vivo folding and physical characterization of an engineered scFv fragment. Protein Eng. 10, 435-444.
Article Disrupting the hydrophobic patches at the antibody variable/...
https://plueckthun.bioc.uzh.ch/wp-content/uploads/Publications/APpub0117.pdf.
While lower overall stability increases the probability that the protein will aggregate, we could find mutations that greatly reduce the aggregation propensity without changing the thermodynamic stability, presumably by selectively destabilising aggregation-prone folding intermediates, thereby altering the kinetic distribution between the folded and the misfolded/aggregated state
Also: Ewert, S., Honegger, A. and Plückthun, A. (2003) Structure-based improvement of the biophysical properties of immunoglobulin VH domains with a generalizable approach. Biochemistry 42, 1517-1528.
Article Structure-Based Improvement of the Biophysical Properties of...
https://plueckthun.bioc.uzh.ch/wp-content/uploads/Publications/APpub0232.pdf
For more on the topic, see https://plueckthun.bioc.uzh.ch/publications/ topic "Antibody engineering"
As you can see in the above publications, we show examples of point mutations that significantly affect the percentage of properly folded product without affecting the stability, as measured by equilibrium denaturation, and other mutations that significantly improved the stability of the native protein, but did not improve the aggregation tendency.
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