18. How can E. coli host cells be engineered so that complex proteins with a large...

18. How can E. coli host cells be engineered so that complex proteins with a large number of disulfide bonds are properly folded and therefore produced in an active form rather than as part of an inclusion body?

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Expert Solution

Some of the available strategies for ensuring formation of properly folded disulfide-bonded proteins in bacteria such as E. coli are -

Periplasmic expression - The periplasm is an oxidizing compartment due to the presence of thioredoxin superfamily proteins and it hosts enzymes catalyzing disulfide bond formation and their isomerization, as well as specific chaperones and foldases. The fusion of recombinant proteins to suitable leader peptides allows for the translocation of unfolded precursors into the periplasm by either the Sec or the SRP system.
Modulation of the expression level - To avoid overcrowding in periplasm, protein secretion can be effectively modulated at the translational level by modifying the Shine-Dalgarno sequence.
By creating double mutant strains of thioredoxin/glutaredoxin reductase (trxB-, gor-) pathway - These strains are characterized by the presence of an oxidizing cytoplasm, compatible with the folding
of proteins that need disulfide bridges for stabilizing their structure.

gladiator answered 1 year ago

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