Cotranslational protein folding studies using Force Profile Analysis, a method where the SecM translational arrest peptide is used to detect folding‐induced forces acting on the nascent polypeptide, ...have so far been limited mainly to small domains of cytosolic proteins that fold in close proximity to the translating ribosome. In this study, we investigate the cotranslational folding of the periplasmic, disulfide bond‐containing Escherichia coli protein alkaline phosphatase (PhoA) in a wild‐type strain background and a strain background devoid of the periplasmic thiol: disulfide interchange protein DsbA. We find that folding‐induced forces can be transmitted via the nascent chain from the periplasm to the polypeptide transferase center in the ribosome, a distance of ~160 Å, and that PhoA appears to fold cotranslationally via at least two disulfide‐stabilized folding intermediates. Thus, Force Profile Analysis can be used to study cotranslational folding of proteins in an extra‐cytosolic compartment, like the periplasm.
We propose a method to compute the
R
-matrix
R
on a tensor product of Fock modules from coproduct relations in a Hopf algebra. We apply this method to the quantum toroidal algebra
U
q
,
t
(
gl
.
.
1
...)
. We show that the coproduct relations of
U
q
,
t
(
gl
.
.
1
)
reduce to a single elegant equation for
R
. Using the theory of symmetric Macdonald polynomials we show that this equation provides a recursive formula for the matrix elements of
R
.
Escherichia coli
has been widely used for the production of recombinant proteins. To improve protein production yields in
E. coli
, directed engineering approaches have been commonly used. However, ...there are only few reported examples of the isolation of
E. coli
protein production strains using evolutionary approaches. Here, we first give an introduction to bacterial evolution and mutagenesis to set the stage for discussing how so far selection- and screening-based approaches have been used to isolate
E. coli
protein production strains. Finally, we discuss how evolutionary approaches may be used in the future to isolate
E. coli
strains with improved protein production characteristics.
We compute the free energy and surface tension function for the
five-vertex model
, a model of non-intersecting monotone lattice paths on the grid in which each corner gets a weight
r
>
0
. We give a ...variational principle for limit shapes in this setting, and show that the resulting Euler–Lagrange equation can be integrated, giving limit shapes explicitly parameterized by analytic functions.
In
, many recombinant proteins are produced in the periplasm. To direct these proteins to this compartment, they are equipped with an N-terminal signal sequence so that they can traverse the ...cytoplasmic membrane via the protein-conducting Sec translocon. Recently, using the single-chain variable antibody fragment BL1, we have shown that harmonizing the target gene expression intensity with the Sec translocon capacity can be used to improve the production yields of a recombinant protein in the periplasm. Here, we have studied the consequences of improving the production of BL1 in the periplasm by using a proteomics approach. When the target gene expression intensity is not harmonized with the Sec translocon capacity, the impaired translocation of secretory proteins, protein misfolding/aggregation in the cytoplasm, and an inefficient energy metabolism result in poor growth and low protein production yields. The harmonization of the target gene expression intensity with the Sec translocon capacity results in normal growth, enhanced protein production yields, and, surprisingly, a composition of the proteome that is-besides the produced target-the same as that of cells with an empty expression vector. Thus, the single-chain variable antibody fragment BL1 can be efficiently produced in the periplasm without causing any notable detrimental effects to the production host. Finally, we show that under the optimized conditions, a small fraction of the target protein is released into the extracellular milieu via outer membrane vesicles. We envisage that our observations can be used to design strategies to further improve the production of secretory recombinant proteins in
The bacterium
is widely used to produce recombinant proteins. Usually, trial-and-error-based screening approaches are used to identify conditions that lead to high recombinant protein production yields. Here, for the production of an antibody fragment in the periplasm of
, we show that an optimization of its production is accompanied by the alleviation of stress. This indicates that the monitoring of stress responses could be used to facilitate enhanced recombinant protein production yields.
The well-established E. coli protein production strain C41(DE3) was isolated from the T7 RNA polymerase-based BL21(DE3) strain for its ability to produce difficult recombinant proteins, and it ...acquired multiple mutations during its isolation. Standard allelic replacement and competition experiments were insufficient to de-convolute these mutations. By reconstructing the evolution of C41(DE3) in real time, we identified the time frames when the different mutations occurred, enabling us to link them to particular stress events. Starvation stress imposed by the isolation procedure selected for mutations enhancing nutrient uptake, and protein production stress for mutations weakening the lacUV5 promoter, which governs t7rnap expression. Moreover, recapitulating protein production stress in BL21(DE3) showed that mutations weakening the lacUV5 promoter occur through RecA-dependent recombination with the wild-type lac-promoter and are selected for upon the production of any protein. Thus, the instability of the lacUV5 promoter in BL21(DE3) alleviates protein production stress and can be harnessed to enhance production.
Display omitted
•Real-time reconstruction of E. coli strain evolution de-convolutes genetic adaptations•E. coli C41(DE3) evolved from BL21(DE3) by nutrient/protein production stress•Weakening PlacUV5 enhances protein production in C41(DE3) by lowering T7 RNAP levels•Any recombinant protein leads to the selection of mutations weakening PlacUV5
Schlegel et al. introduce the concept of reconstructing evolution in real time to de-convolute mutations. This approach was critical to pinpoint the defining mutations that an E. coli strain with improved protein production characteristics had accumulated during its isolation and to elucidate the causes of these mutations.
Reverse genetics (RG) systems have been instrumental for determining the molecular aspects of viral replication, pathogenesis, and for the development of therapeutics. Here, we demonstrate that genes ...encoding the influenza surface antigens hemagglutinin and neuraminidase have varying stability when cloned into a common RG plasmid and transformed into Escherichia coli. Using GFP as a reporter, we demonstrate that E. coli expresses the target genes in the RG plasmid at low levels. Incorporating lac operators or a transcriptional terminator into the plasmid reduced expression and stabilized the viral genes to varying degrees. Sandwiching the viral gene between two lac operators provided the largest contribution to stability and we confirmed the stabilization is Lac repressor-dependent and crucial for subsequent plasmid propagations in E. coli. Viruses rescued from the lac operator-stabilized plasmid displayed similar kinetics and titers to the original plasmid in two different viral backbones. Together, these results indicate that silencing transcription from the plasmid in E. coli helps to maintain the correct influenza gene sequence and that the lac operator addition does not impair virus production. It is envisaged that sandwiching DNA segments between lac operators can be used for reducing DNA segment instability in any plasmid that is propagated in E. coli which express the Lac repressor.
A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used "Walker strains" C41(DE3) ...and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for high-throughput applications.
Many bacteria export effector proteins fulfilling their function in membranes of a eukaryotic host. These effector membrane proteins appear to contain signals for two incompatible bacterial secretion ...pathways in the same protein: a specific export signal, as well as transmembrane segments that one would expect to mediate targeting to the bacterial inner membrane. Here, we show that the transmembrane segments of effector proteins of type III and type IV secretion systems indeed integrate in the membrane as required in the eukaryotic host, but that their hydrophobicity in most instances is just below the threshold required for mediating targeting to the bacterial inner membrane. Furthermore, we show that binding of type III secretion chaperones to both the effector's chaperone-binding domain and adjacent hydrophobic transmembrane segments also prevents erroneous targeting. These results highlight the evolution of a fine discrimination between targeting pathways that is critical for the virulence of many bacterial pathogens.
PDF
