Abstract
BackgroundBacterial 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.ResultsWe 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.ConclusionsIn 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0606-4) contains supplementary material, which is available to authorized users.
Highlights
Bacterial production of natively folded heterologous proteins by secretion to the extracellular space can improve protein production by simplifying purification and enabling continuous processing
We previously reported that the enzyme beta-lactamase adopts a catalytically active conformation after secretion by the type III secretion system (T3SS) [6]
We confirmed that beta-lactamase was active in the extracellular space after secretion by the T3SS, and found that enzymatic activity in the extracellular space was both enzyme- and secretion-dependent (Fig. 1a)
Summary
Bacterial production of natively folded heterologous proteins by secretion to the extracellular space can improve protein production by simplifying purification and enabling continuous processing. 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. The type III secretion system (T3SS) is a protein secretion machine found in Gram-negative pathogenic bacteria. This multimeric heteroprotein structure is characterized by a long passageway that is 2–3 nm in internal diameter, termed the needle [4]. Proteins secreted by a T3SS have been previously shown to adopt a native conformation after secretion, both in the extracellular space and when delivered to the cytoplasm of a neighboring cell [6,7,8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
