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Zhang Z.,University of Stockholm | Kuipers G.,Xbrane Bioscience AB | Niemiec L.,University of Stockholm | Baumgarten T.,University of Stockholm | And 3 more authors.
Microbial Cell Factories | Year: 2015

Background: For membrane protein production, the Escherichia coli T7 RNA polymerase (T7 RNAP)-based protein production strain BL21(DE3) in combination with T7-promoter based expression vectors is widely used. Cells are routinely cultured in Lysogeny broth (LB medium) and expression of the chromosomally localized t7rnap gene is governed by the isopropyl-β-d-1-thiogalactopyranoside (IPTG) inducible lacUV5 promoter. The T7 RNAP drives the expression of the plasmid borne gene encoding the recombinant membrane protein. Production of membrane proteins in the cytoplasmic membrane rather than in inclusion bodies in a misfolded state is usually preferred, but often hampered due to saturation of the capacity of the Sec-translocon, resulting in low yields. Results: Contrary to expectation we observed that omission of IPTG from BL21(DE3) cells cultured in LB medium can lead to significantly higher membrane protein production yields than when IPTG is added. In the complete absence of IPTG cultures stably produce membrane proteins in the cytoplasmic membrane, whereas upon the addition of IPTG membrane proteins aggregate in the cytoplasm and non-producing clones are selected for. Furthermore, in the absence of IPTG, membrane proteins are produced at a lower rate than in the presence of IPTG. These observations indicate that in the absence of IPTG the Sec-translocon capacity is not/hardly saturated, leading to enhanced membrane protein production yields in the cytoplasmic membrane. Importantly, for more than half of the targets tested the yields obtained using un-induced BL21(DE3) cells were higher than the yields obtained in the widely used membrane protein production strains C41(DE3) and C43(DE3). Since most secretory proteins reach the periplasm via the Sec-translocon, we also monitored the production of three secretory recombinant proteins in the periplasm of BL21(DE3) cells in the presence and absence of IPTG. For all three targets tested omitting IPTG led to the highest production levels in the periplasm. Conclusions: Omission of IPTG from BL21(DE3) cells cultured in LB medium provides a very cost- and time effective alternative for the production of membrane and secretory proteins. Therefore, we recommend that this condition is incorporated in membrane- and secretory protein production screens. © 2015 Zhang et al.


Grant
Agency: Cordis | Branch: FP7 | Program: CP-IP | Phase: HEALTH.2011.1.4-4 | Award Amount: 40.88M | Year: 2011

Vaccines so far have been developed mostly by following an empiric approach. To prevent and possibly cure unresolved and emerging infectious diseases we need to fully exploit the potential of the human immune system. Progress in science and technology makes it possible to achieve what was previously deemed impossible. The scope of this project is to produce knowledge necessary to develop novel and powerful immunization technologies for the next generation of human vaccines. This goal requires a multidisciplinary approach in which diverse but complementary scientific disciplines and technologies converge. Therefore some of the most competitive European research groups from public institutions and biotechs have agreed to join forces in ADITEC, together with top US groups on systems biology and adjuvants to support this enterprise. A systems biology approach will be used to study licensed and experimental vaccines in patient characterization studies and in clinical trials, to investigate the effect of adjuvants, vectors, formulations, delivery devices, routes of immunization, homologous and heterologous primeboost schedules, as well as the impact of host factors such as age, gender, genetics and pathologies. Animal models will be used to complement human studies, and to select novel immunization technologies to be advanced to the clinic. To address these issues in a coordinated manner, ADITEC is organised on a matrix structure in which research themes and experimental approaches feed into each other. Training curricula will be created to impact on the formation of the next generation of EU researchers in the field. ADITEC scientists and institutions are part of the Sclavo Vaccines Association (SVA), which is dedicated to vaccines and vaccine research. SVA, acting as the coordinating institution, guarantees the long-term commitment and sustainability of this initiative, beyond the duration of ADITEC itself.


PubMed | University of Groningen, Xbrane Bioscience AB and University of Stockholm
Type: | Journal: Microbial cell factories | Year: 2015

For membrane protein production, the Escherichia coli T7 RNA polymerase (T7 RNAP)-based protein production strain BL21(DE3) in combination with T7-promoter based expression vectors is widely used. Cells are routinely cultured in Lysogeny broth (LB medium) and expression of the chromosomally localized t7rnap gene is governed by the isopropyl--D-1-thiogalactopyranoside (IPTG) inducible lacUV5 promoter. The T7 RNAP drives the expression of the plasmid borne gene encoding the recombinant membrane protein. Production of membrane proteins in the cytoplasmic membrane rather than in inclusion bodies in a misfolded state is usually preferred, but often hampered due to saturation of the capacity of the Sec-translocon, resulting in low yields.Contrary to expectation we observed that omission of IPTG from BL21(DE3) cells cultured in LB medium can lead to significantly higher membrane protein production yields than when IPTG is added. In the complete absence of IPTG cultures stably produce membrane proteins in the cytoplasmic membrane, whereas upon the addition of IPTG membrane proteins aggregate in the cytoplasm and non-producing clones are selected for. Furthermore, in the absence of IPTG, membrane proteins are produced at a lower rate than in the presence of IPTG. These observations indicate that in the absence of IPTG the Sec-translocon capacity is not/hardly saturated, leading to enhanced membrane protein production yields in the cytoplasmic membrane. Importantly, for more than half of the targets tested the yields obtained using un-induced BL21(DE3) cells were higher than the yields obtained in the widely used membrane protein production strains C41(DE3) and C43(DE3). Since most secretory proteins reach the periplasm via the Sec-translocon, we also monitored the production of three secretory recombinant proteins in the periplasm of BL21(DE3) cells in the presence and absence of IPTG. For all three targets tested omitting IPTG led to the highest production levels in the periplasm.Omission of IPTG from BL21(DE3) cells cultured in LB medium provides a very cost- and time effective alternative for the production of membrane and secretory proteins. Therefore, we recommend that this condition is incorporated in membrane- and secretory protein production screens.


Grant
Agency: Cordis | Branch: FP7 | Program: MC-ITN | Phase: FP7-PEOPLE-2013-ITN | Award Amount: 3.42M | Year: 2013

Even in the simplest cells, the integration of proteins into a biological membrane is a complex process that is frequently coupled to ribosomal protein synthesis, and requires the coordinated actions of several additional cellular machines. The de novo recapitulation of such a complex process is well beyond the scope of our current technical abilities. Indeed, the techniques that are used to create novel proteoliposomes, for drug delivery, and artificial membranes, for synthetic biology, are extremely crude. A common approach is to mix detergent solubilised proteins with lipids, and then remove the detergent to form proteoliposomes, a process that is inefficient and difficult to control. Another major limitation of this approach is our inability to alter the protein complement of the resulting phospholipid-bilayers once they are formed. It is precisely this issue that our consortium will address, by creating a flexible and ubiquitous platform that is ideally suited to incorporating proteins into preformed liposomes. To achieve this novel and innovative breakthrough in liposome technology, we will harness the unusual ability of tail-anchored proteins to be inserted into pre-existing membranes. This technique will enable the production of customised liposomes that can be tailored to optimise drug delivery, and allow the creation of multifunctional artificial membranes for the newly emerging field of synthetic biology. The overriding ethos of our network is to develop a robust platform for the application and exploitation of tail-anchored membrane proteins based on a framework that develops and enhances fundamental insight and training in this new field of research.


Daleke-Schermerhorn M.H.,VU University Amsterdam | Daleke-Schermerhorn M.H.,Abera Bioscience AB | Felix T.,Institute Pasteur Paris | Felix T.,French Institute of Health and Medical Research | And 25 more authors.
Applied and Environmental Microbiology | Year: 2014

Outer membrane vesicles (OMVs) are spherical nanoparticles that naturally shed from Gram-negative bacteria. They are rich in immunostimulatory proteins and lipopolysaccharide but do not replicate, which increases their safety profile and renders them attractive vaccine vectors. By packaging foreign polypeptides in OMVs, specific immune responses can be raised toward heterologous antigens in the context of an intrinsic adjuvant. Antigens exposed at the vesicle surface have been suggested to elicit protection superior to that from antigens concealed inside OMVs, but hitherto robust methods for targeting heterologous proteins to the OMV surface have been lacking. We have exploited our previously developed hemoglobin protease (Hbp) autotransporter platform for display of heterologous polypeptides at the OMV surface. One, two, or three of the Mycobacterium tuberculosis antigens ESAT6, Ag85B, and Rv2660c were targeted to the surface of Escherichia coli OMVs upon fusion to Hbp. Furthermore, a hypervesiculating ΔtolR ΔtolA derivative of attenuated Salmonella enterica serovar Typhimurium SL3261 was generated, enabling efficient release and purification of OMVs decorated with multiple heterologous antigens, exemplified by the M. tuberculosis antigens and epitopes from Chlamydia trachomatis major outer membrane protein (MOMP). Also, we showed that delivery of Salmonella OMVs displaying Ag85B to antigen-presenting cells in vitro results in processing and presentation of an epitope that is functionally recognized by Ag85B-specific T cell hybridomas. In conclusion, the Hbp platform mediates efficient display of (multiple) heterologous antigens, individually or combined within one molecule, at the surface of OMVs. Detection of antigen- specific immune responses upon vesicle-mediated delivery demonstrated the potential of our system for vaccine development. © 2014, American Society for Microbiology.


Hjelm A.,University of Stockholm | Soderstrom B.,University of Stockholm | Soderstrom B.,Okinawa Institute of Science and Technology | Vikstrom D.,Xbrane Bioscience AB | And 5 more authors.
Applied and Environmental Microbiology | Year: 2015

Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts aregenerated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlledproduction of the bacteriophage ΦX174 lysis protein E. While ghosts possess all the immunostimulatory surface properties ofthe original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineeredthe Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologousproteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenictarget of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use ofdifferent promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitoredby determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flowcytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a proteaseaccessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concertedproduction of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently displayantigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsicadjuvant activity and exposure of heterologous antigens to the immune system. © 2015, American Society for Microbiology.


PubMed | VU University Amsterdam, Xbrane Bioscience AB and University of Stockholm
Type: Journal Article | Journal: Applied and environmental microbiology | Year: 2015

Bacterial ghosts are empty cell envelopes of Gram-negative bacteria that can be used as vehicles for antigen delivery. Ghosts are generated by releasing the bacterial cytoplasmic contents through a channel in the cell envelope that is created by the controlled production of the bacteriophage X174 lysis protein E. While ghosts possess all the immunostimulatory surface properties of the original host strain, they do not pose any of the infectious threats associated with live vaccines. Recently, we have engineered the Escherichia coli autotransporter hemoglobin protease (Hbp) into a platform for the efficient surface display of heterologous proteins in Gram-negative bacteria, HbpD. Using the Mycobacterium tuberculosis vaccine target ESAT6 (early secreted antigenic target of 6 kDa), we have explored the application of HbpD to decorate E. coli and Salmonella ghosts with antigens. The use of different promoter systems enabled the concerted production of HbpD-ESAT6 and lysis protein E. Ghost formation was monitored by determining lysis efficiency based on CFU, the localization of a set of cellular markers, fluorescence microscopy, flow cytometry, and electron microscopy. Hbp-mediated surface display of ESAT6 was monitored using a combination of a protease accessibility assay, fluorescence microscopy, flow cytometry and (immuno-)electron microscopy. Here, we show that the concerted production of HbpD and lysis protein E in E. coli and Salmonella can be used to produce ghosts that efficiently display antigens on their surface. This system holds promise for the development of safe and cost-effective vaccines with optimal intrinsic adjuvant activity and exposure of heterologous antigens to the immune system.

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