Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

Metcalf, Kevin J.; Finnerty, Casey; Azam, Anum; Valdivia, Elias; Tullman‐Ercek, Danielle

doi:10.1128/aem.01330-14

Cited by 20 publications

(48 citation statements)

References 52 publications

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“…No cofactors are required for activity [14]. We previously reported that the enzyme beta-lactamase adopts a catalytically active conformation after secretion by the T3SS [6]. We confirmed that beta-lactamase was indeed 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.…”

Section: Resultssupporting

confidence: 81%

“…In addition, the same kinetic parameters were calculated for secreted enzyme (Table 1). This analysis was first performed in standard production media (Lysogeny Broth, Lennox; 5 g L −1 NaCl) [6]. …”

Section: Resultsmentioning

confidence: 99%

“…Indeed, cryo-electron microscopy of secretion suggests that proteins are fully linearized before being ejected into the extracellular space [5]. 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 [6–8]. …”

Section: Introductionmentioning

confidence: 99%

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Proteins adopt functionally active conformations after type III secretion

et al. 2016

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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.

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Section: Resultssupporting

confidence: 81%

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Proteins adopt functionally active conformations after type III secretion

et al. 2016

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“…Thus, in addition to the well-studied T3S-associated "classical" N-terminal secretion signals, we identified a C-terminal secretion signal that mediates secretion of both endogenous and exogenous proteins. The ability of T3SS to export proteins directly from the bacterial cytoplasm into the extracellular medium or into the host cell cytosol has been exploited for heterologous protein production or antigen delivery during vaccination (46,47). In this context, the newly identified C-terminal T3S signal may have interesting biotechnological applications for production of proteins with N-terminal region that cannot be modified without affecting the protein functionality.…”

Section: Discussionmentioning

confidence: 99%

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal

Wolf‐Watz

2015

Journal of Biological Chemistry

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Background: After auto-proteolysis and dissociation, YscU CC is secreted by the T3SS of Yersinia pseudotuberculosis. Results: YscU CC harbors a specific C-terminal T3S signal and its deletion triggers an increase of YscF secretion without affecting Yops secretion. Conclusion: C-terminal end of YscU participate in YscF secretion regulation but not in the substrate specificity switch. Significance: This is the first report of a C-terminal T3S signal.

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“…A major level of control of the expression of the SPI-1 and SPI-2 T3SS genes is via the activity of transcriptional regulators, and these regulators can be used to vary the level of SPI-1 and SPI-2 gene expression under various conditions Garmendia et al 2003;Ellermeier and Slauch 2007;Fass and Groisman 2009;Metcalf et al 2014). The expression of SPI-1 and SPI-2 genes from their normal chromosomal locations in the S. Typhimurium genome requires the hilA and ssrAB genes, respectively (Bajaj et al 1995;Ochman et al 1996;Deiwick et al 1999;Garmendia et al 2003;Ellermeier and Slauch 2007;Fass and Groisman 2009).…”

Section: The Role Of Key Transcriptional Regulators In Cloned Spi-1 Amentioning

confidence: 99%

Transfer of the cloned Salmonella SPI-1 type III secretion system and characterization of its expression mechanisms in Gram negative bacteria in comparison with cloned SPI-2

Cangelosi

Hannagan

Santiago

et al. 2015

Microbiological Research

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Cloned type III secretion systems have much potential to be used for bacterial engineering purposes involving protein secretion and substrate translocation directly into eukaryotic cells. We have previously cloned the SPI-1 and SPI-2 type III systems from the Salmonella enterica serovar Typhimurium genome using plasmid R995 which can conveniently capture large genomic segments for transfer between bacterial strains. However, though expressed and functional in Salmonella strains, cloned SPI-1 was previously observed to have a serious expression defect in other Gram negative bacteria including Escherichia coli. Here we show that cloned SPI-1 expression and secretion can be detected in the secretion preps from E. coli and Citrobacter indicating the first observation of non-Salmonella SPI-1 expression. We describe a compatible plasmid system to introduce engineered SPI-1 substrates into cloned SPI-1 strains. However, a SPI-1 translocation defect is still observed in E. coli, and we show that this is likely due to a defect in SipB expression/secretion in this species. In addition, we also examined the requirement for the hilA and ssrAB regulators in the expression of cloned SPI-1 and SPI-2, respectively. We found a strict requirement for hilA for full cloned SPI-1 expression and secretion. However, though we found that ssrAB is required for full cloned SPI-2 expression in a range of media across different bacteria, it is not required for cloned SPI-2 expression in MgM8 inducing media in S. Typhimurium. This suggests that under SPI-2 inducing conditions in S. Typhimurium, other factors can substitute for loss of ssrAB in cloned SPI-2 expression. The results provide key foundational information for the future use of these cloned systems in bacteria.

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Using Transcriptional Control To Increase Titers of Secreted Heterologous Proteins by the Type III Secretion System

Cited by 20 publications

References 52 publications

Proteins adopt functionally active conformations after type III secretion

Proteins adopt functionally active conformations after type III secretion

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal

Transfer of the cloned Salmonella SPI-1 type III secretion system and characterization of its expression mechanisms in Gram negative bacteria in comparison with cloned SPI-2

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