(392e) Investigating the Status of Oxidative Protein Folding in Escherichia Coli Missing Components of the Thiol-Redox Pathways | AIChE

(392e) Investigating the Status of Oxidative Protein Folding in Escherichia Coli Missing Components of the Thiol-Redox Pathways


Veeravalli, K. - Presenter, University of Texas at Austin
Chrysostomou, C. - Presenter, The University of Texas at Austin
Georgiou, G. - Presenter, The University of Texas at Austin
Faulkner, M. - Presenter, Harvard Medical School
Beckwith, J. - Presenter, Harvard Medical School

The thioredoxin and glutaredoxin pathways in the cytoplasm of Escherichia coli are responsible for the maintenance of cytoplasm in a reduced state, thus strongly disfavoring the formation of structural disulfide bonds in cytosolic proteins. E. coli mutants missing components of both these pathways are not viable as essential enzymes like ribonucleotide reductase depends on atleast one of these pathways for the reduction of disulfide bond formed during its catalytic cycle. Such mutants having non functional thioredoxin and glutaredoxin pathways accumulate suppressor mutations in the gene ahpC encoding a peroxiredoxin, which rescues the growth defect. In this study, we explored oxidative protein folding in cytoplasm of E. coli mutants, missing components of both the thioredoxin and glutaredoxin pathways, to determine the optimal conditions for maximizing the expression yield of multiple disulfide-bonded proteins. Our results indicate that the correct folding of proteins with complex patterns of disulfide bonds like tissue plasminogen activator depends on whether the strains can synthesize glutathione or not as well as the nature of the suppressor allele in the peroxiredoxin AhpC to a certain extent. Also, we found that the strains missing thioredoxin reductase and glutathione accumulate high yields of proteins with fewer disulfide bonds like alkaline phosphatase, and antibody fragments. Consequently, the latter strains provide an exciting platform for high level expression of antibody fragments and as a result could be a useful expression host for biotechnological purposes. To understand some of the differences observed among the thiol-redox pathway mutants in supporting disulfide bond formation, a DNA microarray based global transcriptional profiling was performed. While the genes involved in chemotactic responses and flagellar biosynthesis seem to be greatly repressed in the thiol-redox pathway mutant strains, genes involved in oxidative stress reponses, cold shock and envelope stresses are highly upregulated. The results from the transcriptional analysis data are being employed to construct strains that exhibit more extensive disulfide bond formation in the cytoplasm.