Proteins adopt functionally active conformations after type III secretion

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.

Delivery of Heterologous Proteins, Enzymes, and Antigens via the Bacterial Type III Secretion System

“…Once proteins pass through the T3S injectisome, they must refold into the correct conformations. This process has been analyzed in the interest of protein production and isolation [38]. During a typical protein production process, proteins accumulate within the cytosol of bacterial cells or within inclusion bodies within the cytosol.…”

“…Secretion through the T3SS could serve as a useful strategy for protein purification, as the protein of interest can be selectively transported to the supernatant of the bacterial culture, removing it from the bacterial proteome; however, since secreted proteins are necessarily unfolded before T3SS-mediated transport, correct folding of the protein is required in the supernatant. The extent of protein refolding in the extracellular space was quantified by Metcalf et al, who compared the refolding of two different enzymes, β-lactamase EC:3.5.2.6 and the alkaline phosphatase EC 3.1.3.1, after secretion [38].…”

“…The β-lactamase, EC:3.5.2.6, class A, is a monomeric enzyme with one disulfide bond that is not required for enzymatic function [38]. In addition, no cofactors are required for enzymatic activity.…”

“…Alkaline phosphatase (AP) EC 3.1.3.1, isozyme 1, is a dimeric enzyme that requires the acquisition of one Mg 2+ ion and two Zn 2+ ions, as well as the formation of two disulfide bonds for enzymatic activity [38]. Enzymatic activity was observed in secreted samples, indicating that disulfide bond formation had occurred to produce the catalytically active conformation.…”

“…In recent years the secretory function of T3SSs has been explored for delivery of antigens in vaccination and as a tool for the production and secretion of heterologous proteins [36][37][38]. To better understand the applicational capabilities of the T3SS, the expression and secretion of heterologous proteins has been investigated.…”