Proteins adopt functionally active conformations after type III secretion
Posted on 2016-12-23 - 05:00
Abstract Background 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. Results We probed correct folding by assaying the function after secretion of two enzymesâbeta-lactamase and alkaline phosphataseâand one single-chain variable fragment of an antibody. Secreted proteins are correctly folded and functional after unfolding, secretion, and refolding in the extracellular space. Furthermore, structural and chemical features required for protein function, such as multimerization and disulfide bond formation, are evident in the secreted protein samples. Finally, the concentration of NaCl in the culture media affects the folding efficiency of secreted proteins in a protein-specific manner. Conclusions In the extracellular space, secreted proteins are able to fold to active conformations, which entails post-translational modifications including: folding, multimerization, acquisition of metal ion cofactors, and formation of disulfide bonds. Further, different proteins have different propensities to refold in the extracellular space and are sensitive to the chemical environment in the extracellular space. Our results reveal strategies to control the secretion and correct folding of diverse target proteins during bacterial cell culture.
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Metcalf, Kevin; Bevington, James; Rosales, Sandy; Burdette, Lisa; Valdivia, Elias; Tullman-Ercek, Danielle (2016). Proteins adopt functionally active conformations after type III secretion. figshare. Collection. https://doi.org/10.6084/m9.figshare.c.3652445.v1
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- Developmental Biology
- Infectious Diseases
- Computational Biology