Engineering disulfide isomerization – Bardwell Lab

Engineering disulfide isomerization

Exploring protein folding pathways in the cell

The folding pathways of various model proteins are well studied in vitro. However, very little in known about in vivo folding pathways. We can use our biosensors to streamline in vivo folding pathways. For BPTI this eliminated a number of problematic disulfide isomerization intermediates shown shaded in grey.

Foit et al (2011) Genetic Selection for Enhanced Folding In Vivo Targets the Cys14-Cys38 Disulfide Bond in Bovine Pancreatic Trypsin Inhibitor. Antioxid Redox Signal. 2011 Mar 15;14(6):973-84 pdf_icon


Optimization of disulfide isomerization

We have developed biosensors specific for disulfide isomerization and used them to evolve alternative pathways that work in the complete absence of the normal machinery for disulfide isomerization. They substitute the isomerase DsbC with the oxidase DsbA and the chaperone DegP. Our new pathway partially mimics, in E. coli, the mechanism used by eukaryotic protein disulfide isomerase. We are opening up new strategies to select for bacteria with an enhanced capacity to express multi-disulfide proteins of pharmaceutical importance.

Ren, G, and Bardwell, J (2011) Engineered pathways for correct disulfide bond formation. Antioxid Redox Signal. Epub before print 2011 Jan 21. pdf_icon



Evolving one isomerase into another

By reversing divergent evolution, we found that a very small proportion of the numerous sequence and structural differences between two distantly related protein disulfide isomerase proteins are important for their functional differences. Our findings strongly support the surprising conclusion that, for these two proteins at least, regions important in determining functional differences represent only a tiny fraction of overall protein structure and that most of the many structural and sequence differences are functionally neutral. In our case, laboratory evolution appears to have taken a direct route to convert one protein family member into another, bypassing the need for multiple changes taking place over ~0.5 billion years of evolution.

Hiniker et. al. (2007) Laboratory evolution of one disulfide isomerase to resemble another. Proc Natl Acad Sci U S A 104: 11670-11675. pdf_icon HHMI News story.


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