Proteins adopt functionally active conformations after type III secretion

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 enzymes-beta-lactamase and alkaline phosphatase-and 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.

Keywords: Protein folding; Protein secretion; T3SS.

Fig. 1

Secreted proteins adopt functional conformations. Activity or ELISA signal is given for samples analyzed from the culture supernatant. Genetic modifications described are with respect to the mature native protein sequence of the POI in the fusion. All proteins are of the format SptP-POI-2xFLAG-6xHIS. Results are plotted for the POIs. a Beta-lactamase (Bla). b Alkaline phosphatase (PhoA). c Single chain variable fragment against anthrax protective antigen (14B7*). The mean is plotted from three biological replicate experiments and the error bars represent one standard deviation. Western blots are representative of the samples analyzed in the functional assays

Fig. 2

Western blots of secreted fusion protein samples subjected to the selective alkylation procedure separated by SDS-PAGE. All proteins are of the format SptP-POI-2xFLAG-6xHIS. Representative images are presented from a western blot for the POIs. a Bla. b PhoA. c 14B7*

Fig. 3

NaCl concentration in media affects secreted protein titer and folding efficiency. Both proteins studied are in the fusion format SptP-POI-2xFLAG-6xHIS. For all plots, unless specified, the mean of three biological replicates is plotted, except where noted by the symbol * to indicate two biological replicates. Error bars represent one standard deviation, unless noted. a i Plot of raw activity of secreted Bla as a function of growth media with representative western blot of analyzed samples. ii Plot of f _fold for secreted Bla as a function of growth media. iii Plot of K _M for secreted Bla as a function of growth media. Error bars represent the standard error. b. i Plot of raw activity of secreted PhoA as a function of growth media with representative western blot of analyzed samples. ii Plot of f _fold for secreted PhoA as a function of growth media. iii Plot of K _M for secreted PhoA as a function of growth media. Error bars represent the standard error