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Quality and Quantity: Protein Production and Folding Using a Bacterial Protein Secretion System

Authors: 
Rosales, S., University of California, Berkeley
Valdivia, E., University of California, Berkeley
Tullman-Ercek, D., University of California, Berkeley

Two process metrics must be considered when producing a heterologous protein: product titer (i.e., quantity); and activity and folding of the protein (i.e., quality). Protein secretion can increase product titer by simplifying purification steps and enabling a continuous process. We studied the effect of secretion on protein folding to improve product quality. In Gram-negative bacteria, proteins can be secreted in one-step from the cytosol to the extracellular space using the type III secretion system. Recent structural data shows that the protein is unfolded and linearized as it is passes through the secretion needle, which is 2 nm in diameter and 50 nm long. Proteins must thus refold in the extracellular space after secretion for this approach to be viable. Indeed, functional assays show enzymatic activity or antigen binding after secretion of two model enzymes and a single chain variable fragment of an antibody, suggesting that these proteins do fold after secretion. Genetic and chemical methods show these proteins form disulfide bonds and multimerize in the extracellular space after secretion. Finally, we find the fraction of folding is dependent on both the protein of interest and media composition. The ability to recover folded heterologous protein in the extracellular space demonstrates a strength of the use of a protein secretion chassis.