Silencing Transcription from an Influenza Reverse Genetics Plasmid in Enhances Gene Stability.

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 . Using GFP as a reporter, we demonstrate that expresses the target genes in the RG plasmid at low levels.

Strategies to Enhance Periplasmic Recombinant Protein Production Yields in .

Main reasons to produce recombinant proteins in the periplasm of rather than in its cytoplasm are to -i- enable disulfide bond formation, -ii- facilitate protein isolation, -iii- control the nature of the N-terminus of the mature protein, and -iv- minimize exposure to cytoplasmic proteases. However, hampered protein targeting, translocation and folding as well as protein instability can all negatively affect periplasmic protein production yields. Strategies to enhance periplasmic protein production yields have focused on harmonizing secretory recombinant protein production rates with the capacity of the secretory apparatus by transcriptional and translational tuning, signal peptide selection and engineering, increasing the targeting, translocation and periplasmic folding capacity of the production host, preventing proteolysis, and, finally, the natural and engineered adaptation of the production host to periplasmic protein production.

Cotranslational folding of alkaline phosphatase in the periplasm of Escherichia coli.

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.

Can Adapt Its Protein Translocation Machinery for Enhanced Periplasmic Recombinant Protein Production.

Recently, we engineered a tunable rhamnose promoter-based setup for the production of recombinant proteins in . This setup enabled us to show that being able to precisely set the production rate of a secretory recombinant protein is critical to enhance protein production yields in the periplasm. It is assumed that precisely setting the production rate of a secretory recombinant protein is required to harmonize its production rate with the protein translocation capacity of the cell.

Enhancing Recombinant Protein Yields in the Periplasm by Combining Signal Peptide and Production Rate Screening.

Proteins that contain disulfide bonds mainly mature in the oxidative environment of the eukaryotic endoplasmic reticulum or the periplasm of Gram-negative bacteria. In , disulfide bond containing recombinant proteins are often targeted to the periplasm by an N-terminal signal peptide that is removed once it passes through the Sec-translocon in the cytoplasmic membrane. Despite their conserved targeting function, signal peptides can impact recombinant protein production yields in the periplasm, as can the production rate.

Shaping Escherichia coli for recombinant membrane protein production.

The bacterium Escherichia coli has been widely used for the production of both pro- and eukaryotic membrane proteins. Usually, a set of standard strains as well as different culture setups and induction regimes are screened for to enhance production yields. However, on a limited scale, E.

SRP, FtsY, DnaK and YidC Are Required for the Biogenesis of the E. coli Tail-Anchored Membrane Proteins DjlC and Flk.

Tail-anchored membrane proteins (TAMPs) are relatively simple membrane proteins characterized by a single transmembrane domain (TMD) at their C-terminus. Consequently, the hydrophobic TMD, which acts as a subcellular targeting signal, emerges from the ribosome only after termination of translation precluding canonical co-translational targeting and membrane insertion. In contrast to the well-studied eukaryotic TAMPs, surprisingly little is known about the cellular components that facilitate the biogenesis of bacterial TAMPs.

The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli.

Background: To optimize the production of membrane and secretory proteins in Escherichia coli, it is critical to harmonize the expression rates of the genes encoding these proteins with the capacity of their biogenesis machineries. Therefore, we engineered the Lemo21(DE3) strain, which is derived from the T7 RNA polymerase-based BL21(DE3) protein production strain. In Lemo21(DE3), the T7 RNA polymerase activity can be modulated by the controlled co-production of its natural inhibitor T7 lysozyme.

Tailoring Escherichia coli for the l-Rhamnose P Promoter-Based Production of Membrane and Secretory Proteins.

Membrane and secretory protein production in Escherichia coli requires precisely controlled production rates to avoid the deleterious saturation of their biogenesis pathways. On the basis of this requirement, the E. coli l-rhamnose P promoter (PrhaBAD) is often used for membrane and secretory protein production since PrhaBAD is thought to regulate protein production rates in an l-rhamnose concentration-dependent manner.