Dataset Information

Tuning recombinant protein expression to match secretion capacity.

ABSTRACT: BACKGROUND:The secretion of recombinant disulfide-bond containing proteins into the periplasm of Gram-negative bacterial hosts, such as E. coli, has many advantages that can facilitate product isolation, quality and activity. However, the secretion machinery of E. coli has a limited capacity and can become overloaded, leading to cytoplasmic retention of product; which can negatively impact cell viability and biomass accumulation. Fine control over recombinant gene expression offers the potential to avoid this overload by matching expression levels to the host secretion capacity. RESULTS:Here we report the application of the RiboTite gene expression control system to achieve this by finely controlling cellular expression levels. The level of control afforded by this system allows cell viability to be maintained, permitting production of high-quality, active product with enhanced volumetric titres. CONCLUSIONS:The methods and systems reported expand the tools available for the production of disulfide-bond containing proteins, including antibody fragments, in bacterial hosts.

SUBMITTER: Horga LG

PROVIDER: S-EPMC6303999 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Tuning recombinant protein expression to match secretion capacity.

Horga Luminita Gabriela LG Halliwell Samantha S Castiñeiras Tania Selas TS Wyre Chris C Matos Cristina F R O CFRO Yovcheva Daniela S DS Kent Ross R Morra Rosa R Williams Steven G SG Smith Daniel C DC Dixon Neil N

Microbial cell factories 20181222 1

<h4>Background</h4>The secretion of recombinant disulfide-bond containing proteins into the periplasm of Gram-negative bacterial hosts, such as E. coli, has many advantages that can facilitate product isolation, quality and activity. However, the secretion machinery of E. coli has a limited capacity and can become overloaded, leading to cytoplasmic retention of product; which can negatively impact cell viability and biomass accumulation. Fine control over recombinant gene expression offers the p ...[more]

PMID: 30577801

Similar Datasets

Project description:BackgroundMany valuable biopharmaceutical and biotechnological proteins have been produced in Escherichia coli, however these proteins are almost exclusively localised in the cytoplasm or periplasm. This presents challenges for purification, i.e. the removal of contaminating cellular constituents. One solution is secretion directly into the surrounding media, which we achieved via the 'hijack' of the flagellar type III secretion system (FT3SS). Ordinarily flagellar subunits are exported through the centre of the growing flagellum, before assembly at the tip. However, we exploit the fact that in the absence of certain flagellar components (e.g. cap proteins), monomeric flagellar proteins are secreted into the supernatant.ResultsWe report the creation and iterative improvement of an E. coli strain, by means of a modified FT3SS and a modular plasmid system, for secretion of exemplar proteins. We show that removal of the flagellin and HAP proteins (FliC and FlgKL) resulted in an optimal prototype. We next developed a high-throughput enzymatic secretion assay based on cutinase. This indicated that removal of the flagellar motor proteins, motAB (to reduce metabolic burden) and protein degradation machinery, clpX (to boost FT3SS levels intracellularly), result in high capacity secretion. We also show that a secretion construct comprising the 5'UTR and first 47 amino acidsof FliC from E. coli (but no 3'UTR) achieved the highest levels of secretion. Upon combination, we show a 24-fold improvement in secretion of a heterologous (cutinase) enzyme over the original strain. This improved strain could export a range of pharmaceutically relevant heterologous proteins [hGH, TrxA, ScFv (CH2)], achieving secreted yields of up to 0.29 mg L-1, in low cell density culture.ConclusionsWe have engineered an E. coli which secretes a range of recombinant proteins, through the FT3SS, to the extracellular media. With further developments, including cell culture process strategies, we envision further improvement to the secreted titre of recombinant protein, with the potential application for protein production for biotechnological purposes.

Project description:Investigation of whole genome gene expression level changes in Ashbya gossypii VTT D-101398 secreting recombinant endoglucanase I (EGI) from Trichoderma ressei (Ribeiro et al. 2010 - PMID: 20422178), compared to the its corresponding empty vector control strain and to conditions where VTT D-101398 EGI secreting cultures were treated with dithiothreitol (DTT). Background: Ashbya gossypii is a filamentous Saccharomycete used for the industrial production of riboflavin that has been recently explored as a host system for recombinant protein production. To gain insight into the protein secretory pathway of this biotechnologically relevant fungus, we undertook genome-wide analyses to explore its secretome and its transcriptional responses to protein secretion stress. Results: A computational pipeline was used to predict the inventory of proteins putatively secreted by A. gossypii via the general secretory pathway. The proteins actually secreted by this fungus into the supernatants of submerged cultures in minimal and rich medium were mapped by two-dimensional gel electrophoresis, revealing that most of the A. gossypii secreted proteins have an isoelectric point between 4 and 6, and a molecular mass above 25 kDa. These analyses together indicated that 1-4% of A. gossypii proteins are likely to be secreted, of which less than 33% are putative hydrolases. Furthermore, transcriptomic analyses carried out in A. gossypii cells under recombinant protein secretion conditions and dithiothreitol-induced secretion stress unexpectedly revealed that a conventional unfolded protein response (UPR) was not activated in any of the conditions, as the expression levels of several well-known UPR target genes (e.g. IRE1, KAR2, HAC1 and PDI1 homologs) remained unaffected. However, several other genes involved in protein unfolding, endoplasmatic reticulum-associated degradation, proteolysis, vesicle trafficking, vacuolar protein sorting, secretion and mRNA degradation were up-regulated by dithiothreitol-induced secretion stress. Conversely, the transcription of several genes encoding secretory proteins, such as components of the glycosylation pathway, was severely repressed by dithiothreitol Conclusions: This study provides the first insights into the secretion stress response of A. gossypii, as well as a basic understanding of its protein secretion potential, which is more similar to that of yeast than to that of other filamentous fungi. Contrary to what has been widely described for yeast and fungi, a conventional UPR was not observed in A. gossypii, but alternative protein quality control mechanisms enabled it to cope with secretion stress. These data will help provide strategies for improving heterologous protein secretion in A. gossypii.

Project description:BackgroundProtein secretion in bacteria is an attractive strategy for heterologous protein production because it retains the high titers and tractability of bacterial hosts while simplifying downstream processing. Traditional intracellular production strategies require cell lysis and separation of the protein product from the chemically similar cellular contents, often a multi-step process that can include an expensive refolding step. The type III secretion system of Salmonella enterica Typhimurium transports proteins from the cytoplasm to the extracellular environment in a single step and is thus a promising solution for protein secretion in bacteria. Product titer is sensitive to extracellular environmental conditions, however, and T3SS regulation is integrated with essential cellular functions. Instead of attempting to untangle a complex web of regulatory input, we took an "outside-in" approach to elucidate the effect of growth medium components on secretion titer.ResultsWe dissected the individual and combined effects of carbon sources, buffers, and salts in a rich nutrient base on secretion titer. Carbon sources alone decreased secretion titer, secretion titer increased with salt concentration, and the combination of a carbon source, buffer, and high salt concentration had a synergistic effect on secretion titer. Transcriptional activity measured by flow cytometry showed that medium composition affected secretion system activity, and prolonged secretion system activation correlated strongly with increased secretion titer. We found that an optimal combination of glycerol, phosphate, and sodium chloride provided at least a fourfold increase in secretion titer for a variety of proteins. Further, the increase in secretion titer provided by the optimized medium was additive with strain enhancements.ConclusionsWe leveraged the sensitivity of the type III secretion system to the extracellular environment to increase heterologous protein secretion titer. Our results suggest that maximizing secretion titer via the type III secretion system is not as simple as maximizing secreted protein expression-one must also optimize secretion system activity. This work advances the type III secretion system as a platform for heterologous protein secretion in bacteria and will form a basis for future engineering efforts.

Dataset Information

Tuning recombinant protein expression to match secretion capacity.

Publications

Tuning recombinant protein expression to match secretion capacity.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets