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Proteins adopt functionally active conformations after type III secretion

Metcalf, KJ;Bevington, JL;Rosales, SL;Burdette, LA;Valdivia, E;Tullman-Ercek, D;

Bacterial production of natively folded heterologous proteins by secretion to the extracellular space can improve protein production by simplifying purification and enabling continuous processing. In a typical bacterial protein production process, the protein of interest accumulates in the cytoplasm of the cell, requiring cellular lysis and extensive purification to separate the desired protein from other cellular constituents. The type III secretion system of Gram-negative bacteria is used to secrete proteins from the cytosol to the extracellular space in one step, but proteins must unfold during translocation, necessitating the folding of secreted proteins in the extracellular space for an efficient production process. We evaluated type III secretion as a protein production strategy by characterizing and quantifying the extent of correct folding after secretion.