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  • 1.
    Baumgarten, Thomas
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schlegel, Susan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wagner, Samuel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Löw, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Eriksson, Jonas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bonde, Ida
    Herrgård, Markus J.
    Heipieper, Hermann J.
    Nørholm, Morten H. H.
    Slotboom, Dirk Jan
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, article id 45089Article in journal (Refereed)
    Abstract [en]

    Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production.

  • 2.
    Baumgarten, Thomas
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ytterberg, A. Jimmy
    Zubarev, Roman A.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Optimizing Recombinant Protein Production in the Escherichia coli Periplasm Alleviates Stress2018In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 84, no 12, article id e00270Article in journal (Refereed)
    Abstract [en]

    In Escherichia coli, 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 E. coli.

    IMPORTANCE The bacterium Escherichia coli 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 E. coli, 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.

  • 3. Claassens, Nico J.
    et al.
    Finger-Bou, Max
    Scholten, Bart
    Muis, Frederieke
    de Groot, Jonas J.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Vos, Willem M.
    van der Oost, John
    Bicistronic Design-Based Continuous and High-Level Membrane Protein Production in Escherichia coil2019In: ACS Synthetic Biology, E-ISSN 2161-5063, Vol. 8, no 7, p. 1685-1690Article in journal (Refereed)
    Abstract [en]

    Escherichia coli has been widely used as a platform microorganism for both membrane protein production and cell factory engineering. The current methods to produce membrane proteins in this organism require the induction of target gene expression and often result in unstable, low yields. Here, we present a method combining a constitutive promoter with a library of bicistronic design (BCD) elements, which enables inducer-free, tuned translation initiation for optimal protein production. Our system mediates stable, constitutive production of bacterial membrane proteins at yields that outperform those obtained with E. coli Lemo21(DE3), the current gold standard for bacterial membrane protein production. We envisage that the continuous, fine-tunable, and high-level production of membrane proteins by our method will greatly facilitate their study and their utilization in engineering cell factories.

  • 4. Daleke-Schermerhorn, Maria H.
    et al.
    Felix, Tristan
    Soprova, Zora
    ten Hagen-Jongman, Corinne M.
    Vikström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Xbrane Bioscience AB, Sweden.
    Majlessi, Laleh
    Beskers, Joep
    Follmann, Frank
    de Punder, Karin
    van der Wel, Nicole N.
    Baumgarten, Thomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pham, Thang V.
    Piersma, Sander R.
    Jimenez, Connie R.
    van Ulsen, Peter
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Leclerc, Claude
    Jong, Wouter S. P.
    Luirink, Joen
    Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach2014In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 80, no 18, p. 5854-5865Article in journal (Refereed)
    Abstract [en]

    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 Delta tolR Delta tolA 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.

  • 5.
    Hjelm, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karyolaimos, Alexandros
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zhang, Zhe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rujas, Edurne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vikström, David
    Slotboom, Dirk Jan
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tailoring Escherichia coli for the L-Rhamnose P-BAD Promoter-Based Production of Membrane and Secretory Proteins2017In: ACS Photonics, E-ISSN 2330-4022, Vol. 6, no 6, p. 985-994Article in journal (Refereed)
    Abstract [en]

    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 PBAD 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. By monitoring protein production in real-time in E. coli wild-type and an L-rhamnose catabolism deficient mutant, we demonstrate that the L-rhamnose concentration-dependent tunability of PrhaBAD-mediated protein production is actually due to L-rhamnose consumption rather than regulating production rates. Using this information, a RhaT-mediated L-rhamnose transport and L-rhamnose catabolism deficient double mutant was constructed. We show that this mutant enables the regulation of PrhaBAD-based protein production rates in an L-rhamnose concentration-dependent manner and that this is critical to optimize membrane and secretory protein production yields. The high precision of protein production rates provided by the PrhaBAD promoter in an L-rhamnose transport and catabolism deficient background could also benefit other applications in synthetic biology.

  • 6.
    Hjelm, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schlegel, Susan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Baumgarten, Thomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Klepsch, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wickström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Drew, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Optimizing E. coli-Based Membrane Protein Production Using Lemo21(DE3) and GFP-Fusions2013In: Membrane Biogenesis: Methods and Protocols / [ed] Doron Rapaport, Johannes M. Herrmann, Totowa, USA: Humana Press, 2013, p. 381-400Chapter in book (Refereed)
    Abstract [en]

    Optimizing the conditions for the overexpression of membrane proteins in E. coli and their subsequent purification is usually a laborious and time-consuming process. Combining the Lemo21(DE3) strain, which conveniently allows to identify the optimal expression intensity of a membrane protein using only one strain, and membrane proteins C-terminally fused to Green Fluorescent Protein (GFP) greatly facilitates the production of high-quality membrane protein material for functional and structural studies.

  • 7.
    Hjelm, Anna
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Söderström, Bill
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vikström, David
    Jong, Wouter S. P.
    Luirink, Joen
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Autotransporter-Based Antigen Display in Bacterial Ghosts2015In: Applied and Environmental Microbiology, ISSN 0099-2240, E-ISSN 1098-5336, Vol. 81, no 2, p. 726-735Article in journal (Refereed)
    Abstract [en]

    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 phi 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.

  • 8. Jong, Wouter S. P.
    et al.
    Daleke-Schermerhorn, Maria H.
    Vikström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Xbrane Bioscience AB, Sweden.
    ten Hagen-Jongman, Corinne M.
    de Punder, Karin
    van der Wel, Nicole N.
    van de Sandt, Carolien E.
    Rimmelzwaan, Guus F.
    Follmann, Frank
    Agger, Else Marie
    Andersen, Peter
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Xbrane Bioscience AB, Sweden.
    Luirink, Joen
    An autotransporter display platform for the development of multivalent recombinant bacterial vector vaccines2014In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 13, p. -162Article in journal (Refereed)
    Abstract [en]

    Background: The Autotransporter pathway, ubiquitous in Gram-negative bacteria, allows the efficient secretion of large passenger proteins via a relatively simple mechanism. Capitalizing on its crystal structure, we have engineered the Escherichia coli autotransporter Hemoglobin protease (Hbp) into a versatile platform for secretion and surface display of multiple heterologous proteins in one carrier molecule. Results: As proof-of-concept, we demonstrate efficient secretion and high-density display of the sizeable Mycobacterium tuberculosis antigens ESAT6, Ag85B and Rv2660c in E. coli simultaneously. Furthermore, we show stable multivalent display of these antigens in an attenuated Salmonella Typhimurium strain upon chromosomal integration. To emphasize the versatility of the Hbp platform, we also demonstrate efficient expression of multiple sizeable antigenic fragments from Chlamydia trachomatis and the influenza A virus at the Salmonella cell surface. Conclusions: The successful efficient cell surface display of multiple antigens from various pathogenic organisms highlights the potential of Hbp as a universal platform for the development of multivalent recombinant bacterial vector vaccines.

  • 9. Jong, Wouter S. P.
    et al.
    Vikström, David
    Houben, Diane
    van den Berg van Saparoea, H. Bart
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Luirink, Joen
    Application of an E. coli signal sequence as a versatile inclusion body tag2017In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 16, article id 50Article in journal (Refereed)
    Abstract [en]

    Background: Heterologous protein production in Escherichia coli often suffers from bottlenecks such as proteolytic degradation, complex purification procedures and toxicity towards the expression host. Production of proteins in an insoluble form in inclusion bodies (IBs) can alleviate these problems. Unfortunately, the propensity of heterologous proteins to form IBs is variable and difficult to predict. Hence, fusing the target protein to an aggregation prone polypeptide or IB-tag is a useful strategy to produce difficult-to-express proteins in an insoluble form. Results: When screening for signal sequences that mediate optimal targeting of heterologous proteins to the periplasmic space of E. coli, we observed that fusion to the 39 amino acid signal sequence of E. coli TorA (ssTorA) did not promote targeting but rather directed high-level expression of the human proteins hEGF, Pla2 and IL-3 in IBs. Further analysis revealed that ssTorA even mediated IB formation of the highly soluble endogenous E. coli proteins TrxA and MBP. The ssTorA also induced aggregation when fused to the C-terminus of target proteins and appeared functional as IB-tag in E. coli K-12 as well as B strains. An additive effect on IB-formation was observed upon fusion of multiple ssTorA sequences in tandem, provoking almost complete aggregation of TrxA and MBP. The ssTorA-moiety was successfully used to produce the intrinsically unstable hEGF and the toxic fusion partner SymE, demonstrating its applicability as an IB-tag for difficult-to-express and toxic proteins. Conclusions: We present proof-of-concept for the use of ssTorA as a small, versatile tag for robust E. coli-based expression of heterologous proteins in IBs.

  • 10. Jong, Wouter Sp
    et al.
    Soprova, Zora
    de Punder, Karin
    Ten Hagen-Jongman, Corinne M
    Wagner, Samuel
    Wickström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Andersen, Peter
    van der Wel, Nicole N
    Luirink, Joen
    A structurally informed autotransporter platform for efficient heterologous protein secretion and display.2012In: Microbial cell factories, ISSN 1475-2859, Vol. 11, p. 85-(11 pp.)Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: The self-sufficient autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a β-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein.

    RESULTS: Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development.

    CONCLUSIONS: We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.

  • 11.
    Karyolaimos, Alexandros
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ampah-Korsah, Henry
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hillenaar, Tamara
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Borras, Anna Mestre
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Dolata, Katarzyna Magdalena
    Sievers, Susanne
    Riedel, Katharina
    Daniels, Robert
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Enhancing Recombinant Protein Yields in the E. coli Periplasm by Combining Signal Peptide and Production Rate Screening2019In: Frontiers in Microbiology, ISSN 1664-302X, E-ISSN 1664-302X, Vol. 10, article id 1511Article in journal (Refereed)
    Abstract [en]

    Proteins that contain disulfide bonds mainly mature in the oxidative environment of the eukaryotic endoplasmic reticulum or the periplasm of Gram-negative bacteria. In E. coli, 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 Sectranslocon 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. Here, we present a combined screen involving different signal peptides and varying production rates that enabled the identification of more optimal conditions for periplasmic production of recombinant proteins with disulfide bonds. The data was generated from two targets, a single chain antibody fragment (BL1) and human growth hormone (hGH), with four different signal peptides and a titratable rhamnose promoter-based system that enables the tuning of protein production rates. Across the screen conditions, the yields for both targets significantly varied, and the optimal signal peptide and rhamnose concentration differed for each protein. Under the optimal conditions, the periplasmic BL1 and hGH were properly folded and active. Our study underpins the importance of combinatorial screening approaches for addressing the requirements associated with the production of a recombinant protein in the periplasm.

  • 12.
    Karyolaimos, Alexandros
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ampah-Korsah, Henry
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zhang, Zhe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Shaping Escherichia coli for recombinant membrane protein production2018In: FEMS Microbiology Letters, ISSN 0378-1097, E-ISSN 1574-6968, Vol. 365, no 15, article id fny152Article, review/survey (Refereed)
    Abstract [en]

    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. coli strains have been isolated for recombinant helical bundle membrane protein production using both selection- and engineering-based approaches. Here, we discuss how such approaches have been used so far to shape E. coli for the production of these recombinant membrane proteins and may be used in the future to further enhance production yields.

  • 13.
    Klepsch, Mirjam
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Escherichia coli peptide binding protein OppA has a preference for positively chargedpeptidesManuscript (preprint) (Other academic)
  • 14.
    Klepsch, Mirjam
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kovermann, M.
    Low, C.
    Balbach, J.
    Permentier, H. P.
    Fusetti, F.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Slotboom, D. J.
    Berntsson, R. P. -A
    Escherichia coli Peptide Binding Protein OppA Has a Preference for Positively Charged Peptides2011In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 414, no 1, p. 75-85Article in journal (Refereed)
    Abstract [en]

    The Escherichia coli peptide binding protein OppA is an essential component of the oligopeptide transporter Opp. Based on studies on its orthologue from Salmonella typhimurium, it has been proposed that OppA binds peptides between two and five amino acids long, with no apparent sequence selectivity. Here, we studied peptide binding to E. coli OppA directly and show that the protein has an unexpected preference for basic peptides. OppA was expressed in the periplasm, where it bound to available peptides. The protein was purified in complex with tightly bound peptides. The crystal structure (up to 2.0 angstrom) of OppA liganded with the peptides indicated that the protein has a preference for peptides containing a lysine. Mass spectrometry analysis of the bound peptides showed that peptides between two and five amino acids long bind to the protein and indeed hinted at a preference for positively charged peptides. The preference of OppA for peptides with basic residues, in particular lysines, was corroborated by binding studies with peptides of defined sequence using isothermal titration calorimetry and intrinsic protein fluorescence titration. The protein bound tripeptides and tetrapeptides containing positively charged residues with high affinity, whereas related peptides without lysines/arginines were bound with low affinity. A structure of OppA in an open conformation in the absence of ligands was also determined to 2.0 angstrom, revealing that the initial binding site displays a negative surface charge, consistent with the observed preference for positively charged peptides. Taken together, E. coli OppA appears to have a preference for basic peptides.

  • 15.
    Klepsch, Mirjam M.
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Persson, Jan O.
    Stockholm University, Faculty of Science, Department of Mathematics.
    de Gier, Jan-Willem L.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Consequences of the overexpression of a eukaryotic membrane protein, the human KDEL receptor, in Escherichia coli2011In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 407, no 4, p. 532-542Article in journal (Refereed)
    Abstract [en]

    Escherichia coli is the most widely used host for producing membrane proteins. Thus far, to study the consequences of membrane protein overexpression in E. coli, we have focussed on prokaryotic membrane proteins as overexpression targets. Their overexpression results in the saturation of the Sec translocon, which is a protein-conducting channel in the cytoplasmic membrane that mediates both protein translocation and insertion. Saturation of the Sec translocon leads to (i) protein misfolding/aggregation in the cytoplasm, (ii) impaired respiration, and (iii) activation of the Arc response, which leads to inefficient ATP production and the formation of acetate. The overexpression yields of eukaryotic membrane proteins in E. coli are usually much lower than those of prokaryotic ones. This may be due to differences between the consequences of the overexpression of prokaryotic and eukaryotic membrane proteins in E. coli. Therefore, we have now also studied in detail how the overexpression of a eukaryotic membrane protein, the human KDEL receptor, affects E. coli. Surprisingly, the consequences of the overexpression of a prokaryotic and a eukaryotic membrane protein are very similar. Strain engineering and likely also protein engineering can be used to remedy the saturation of the Sec translocon upon overexpression of both prokaryotic and eukaryotic membrane proteins in E. coli.

  • 16.
    Klepsch, Mirjam
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schlegel, Susan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Evolution of theC41(DE3) and C43(DE3) strainsManuscript (preprint) (Other academic)
  • 17.
    Klepsch, Mirjam
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schlegel, Susan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wickström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Friso, Giulia
    Cornell University, Department of Plant Biology.
    van Wijk, Klaas J.
    Cornell University, Department of Plant Biology.
    Persson, Jan-Olov
    Stockholm University, Faculty of Science, Department of Mathematics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wagner, Samuel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Immobilization of the first dimension in 2D blue native/SDS-PAGE allows the relative quantification of membrane proteomes2008In: Methods, ISSN 1095-9130, Vol. 46, no 2, p. 48-53Article in journal (Refereed)
    Abstract [en]

    In biological membranes many proteins are organized in complexes. The method of choice for the global analysis of the subunits of these complexes is two-dimensional blue native (2D BN)/SDS–PAGE. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS–PAGE. In the currently available protocols the 1st dimension BN gel lanes get distorted during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, rendering them unsuitable for comparative analysis. We have developed a 2D BN/SDS–PAGE protocol where the 1st dimension BN gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN gel lanes, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. 2D BN/SDS–PAGE with an immobilized 1st dimension was used for the comparative analysis of the cytoplasmic membrane proteomes of Escherichia coli cells overexpressing a membrane protein and to create a 2D BN/SDS–PAGE reference map of the E. coli cytoplasmic membrane proteome with 143 identified proteins from 165 different protein spots.

  • 18. Krampen, Lea
    et al.
    Malmsheimer, Silke
    Grin, Iwan
    Trunk, Thomas
    Lührmann, Anja
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wagner, Samuel
    Revealing the mechanisms of membrane protein export by virulence-associated bacterial secretion systems2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 3467Article in journal (Refereed)
    Abstract [en]

    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.

  • 19.
    Kuipers, Grietje
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Xbrane Biopharma AB, Sweden.
    Karyolaimos, Alexandros
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Zhang, Zhe
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ismail, Nurzian
    Trinco, Gianluca
    Vikström, David
    Slotboom, Dirk Jan
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    The tunable pReX expression vector enables optimizing the T7-based production of membrane and secretory proteins in E. coli2017In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 16, article id 226Article in journal (Refereed)
    Abstract [en]

    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. This setup enables to precisely tune target gene expression rates in Lemo21(DE3). The t7lys gene is expressed from the pLemo plasmid using the titratable rhamnose promoter. A disadvantage of the Lemo21(DE3) setup is that the system is based on two plasmids, a T7 expression vector and pLemo. The aim of this study was to simplify the Lemo21(DE3) setup by incorporating the key elements of pLemo in a standard T7-based expression vector.

    Results: By incorporating the gene encoding the T7 lysozyme under control of the rhamnose promoter in a standard T7-based expression vector, pReX was created (ReX stands for Regulated gene eXpression). For two model membrane proteins and a model secretory protein we show that the optimized production yields obtained with the pReX expression vector in BL21(DE3) are similar to the ones obtained with Lemo21(DE3) using a standard T7 expression vector. For another secretory protein, a c-type cytochrome, we show that pReX, in contrast to Lemo21(DE3), enables the use of a helper plasmid that is required for the maturation and hence the production of this heme c protein.

    Conclusions: Here, we created pReX, a T7-based expression vector that contains the gene encoding the T7 lysozyme under control of the rhamnose promoter. pReX enables regulated T7-based target gene expression using only one plasmid. We show that with pReX the production of membrane and secretory proteins can be readily optimized. Importantly, pReX facilitates the use of helper plasmids. Furthermore, the use of pReX is not restricted to BL21(DE3), but it can in principle be used in any T7 RNAP-based strain. Thus, pReX is a versatile alternative to Lemo21(DE3).

  • 20. Lee, Chiara
    et al.
    Kang, Hae Joo
    Hjelm, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Qureshi, Abdul Aziz
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nji, Emmanuel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Choudhury, Hassanul
    Beis, Konstantinos
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Drew, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics. Imperial College London, England.
    MemStar: A one-shot Escherichia coli-based approach for high-level bacterial membrane protein production2014In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 588, no 20, p. 3761-3769Article in journal (Refereed)
    Abstract [en]

    Optimising membrane protein production yields in Escherichia coli can be time- and resource-consuming. Here, we present a simple and effective Membrane protein Single shot amplification recipe: MemStar. This one-shot amplification recipe is based on the E. coli strain Lemo21(DE3), the PASM-5052 auto-induction medium and, contradictorily, an IPTG induction step. Using MemStar, production yields for most bacterial membrane proteins tested were improved to reach an average of 5 mg L-1 per OD600 unit, which is significantly higher than yields obtained with other common production strategies. With MemStar, we have been able to obtain new structural information for several transporters, including the sodium/proton antiporter NapA. (C) 2014 Federation of European Biochemical Societies.

  • 21. Luirink, Joen
    et al.
    Yu, Zhong
    Wagner, Samuel
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biogenesis of inner membrane proteins in Escherichia coli2012In: Biochimica et Biophysica Acta - Bioenergetics, ISSN 0005-2728, E-ISSN 1879-2650, Vol. 1817, no 6, p. 965-976Article, review/survey (Refereed)
    Abstract [en]

    The inner membrane proteome of the model organism Escherichia coli is composed of inner membrane proteins, lipoproteins and peripherally attached soluble proteins. Our knowledge of the biogenesis of inner membrane proteins is rapidly increasing. This is in particular true for the early steps of biogenesis protein targeting to and insertion into the membrane. However, our knowledge of inner membrane protein folding and quality control is still fragmentary. Furthering our knowledge in these areas will bring us closer to understand the biogenesis of individual inner membrane proteins in the context of the biogenesis of the inner membrane proteome of Escherichia coli as a whole. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

  • 22. Peschke, Markus
    et al.
    Le Goff, Mélanie
    Koningstein, Gregory M.
    Karyolaimos, Alexandros
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    van Ulsen, Peter
    Luirink, Joen
    SRP, FtsY, DnaK and YidC Are Required for the Biogenesis of the E. coli Tail-Anchored Membrane Proteins DjIC and Flk2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 3, p. 389-403Article in journal (Refereed)
    Abstract [en]

    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. In this study, we identify DjIC and Flk as bona fide Escherichia co/iTAMPs and show that their TMDs are necessary and sufficient for authentic membrane targeting of the fluorescent reporter mNeonGreen. Using strains conditional for the expression of known E. coli membrane targeting and insertion factors, we demonstrate that the signal recognition particle (SRP), its receptor FtsY, the chaperone DnaK and insertase YidC are each required for efficient membrane localization of both TAMPs. A close association between the TMD of DjIC and Flk with both the Ffh subunit of SRP and YidC was confirmed by site-directed in vivo photo-crosslinking. In addition, our data suggest that the hydrophobicity of the TMD correlates with the dependency on SRP for efficient targeting.

  • 23. Pop, Ovidiu I
    et al.
    Soprova, Zora
    Koningstein, Gregory
    Scheffers, Dirk-Jan
    van Ulsen, Peter
    Wickström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Luirink, Joen
    YidC is required for the assembly of the MscL homopentameric pore.2009In: The FEBS journal, ISSN 1742-4658, Vol. 276, no 17, p. 4891-9Article in journal (Refereed)
    Abstract [en]

    The mechanosensitive channel with large conductance (MscL) of Escherichia coli is formed by a homopentameric assembly of MscL proteins. Here, we describe MscL biogenesis as determined using in vivo approaches. Evidence is presented that MscL is targeted to the inner membrane via the signal recognition particle (SRP) pathway, and is inserted into the lipid bilayer independently of the Sec machinery. This is consistent with published data. Surprisingly, and in conflict with earlier data, YidC is not critical for membrane insertion of MscL. In the absence of YidC, assembly of the homopentameric MscL complex was strongly reduced, suggesting a late role for YidC in the biogenesis of MscL. The data are consistent with the view that YidC functions as a membrane-based chaperone 'module' to facilitate assembly of a subset of protein complexes in the inner membrane of E. coli.

  • 24.
    Rapp, Mikaela
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Drew, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Daley, Daniel O.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Nilsson, Johan
    Carvalho, Tiago
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Melén, Karin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    von Heijne, Gunnar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Experimentally based topology models for E. coli inner membrane proteins2004In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 13, no 4, p. 937-945Article in journal (Refereed)
    Abstract [en]

    Membrane protein topology predictions can be markedly improved by the inclusion of even very limited experimental information. We have recently introduced an approach for the production of reliable topology models based on a combination of experimental determination of the location (cytoplasmic or periplasmic) of a protein's C terminus and topology prediction. Here, we show that determination of the location of a protein's C terminus, rather than some internal loop, is the best strategy for large-scale topology mapping studies. We further report experimentally based topology models for 31 Escherichia coli inner membrane proteins, using methodology suitable for genome-scale studies.

  • 25. Rempel, S.
    et al.
    Colucci, E.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Guskov, A.
    Slotboom, D. J.
    Cysteine-mediated decyanation of vitamin B12 by the predicted membrane transporter BtuM2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, article id 3038Article in journal (Refereed)
    Abstract [en]

    Uptake of vitamin B12 is essential for many prokaryotes, but in most cases the membrane proteins involved are yet to be identified. We present the biochemical characterization and high-resolution crystal structure of BtuM, a predicted bacterial vitamin B12 uptake system. BtuM binds vitamin B12 in its base-off conformation, with a cysteine residue as axial ligand of the corrin cobalt ion. Spectroscopic analysis indicates that the unusual thiolate coordination allows for decyanation of vitamin B12. Chemical modification of the substrate is a property other characterized vitamin B12-transport proteins do not exhibit.

  • 26. Santos, Joana A.
    et al.
    Rempel, Stephan
    Mous, Sandra T. M.
    Pereira, Cristiane T.
    ter Beek, Josy
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Guskov, Albert
    Slotboom, Dirk J.
    Functional and structural characterization of an ECF-type ABC transporter for vitamin B122018In: eLIFE, E-ISSN 2050-084X, Vol. 7, article id e35828Article in journal (Refereed)
    Abstract [en]

    Vitamin B12 (cobalamin) is the most complex B-type vitamin and is synthetized exclusively in a limited number of prokaryotes. Its biologically active variants contain rare organometallic bonds, which are used by enzymes in a variety of central metabolic pathways such as L-methionine synthesis and ribonucleotide reduction. Although its biosynthesis and role as cofactor are well understood, knowledge about uptake of cobalamin by prokaryotic auxotrophs is scarce. Here, we characterize a cobalamin-specific ECF-type ABC transporter from Lactobacillus delbrueckii, ECF-CbrT, and demonstrate that it mediates the specific, ATP-dependent uptake of cobalamin. We solved the crystal structure of ECF-CbrT in an apo conformation to 3.4 angstrom resolution. Comparison with the ECF transporter for folate (ECF-FoIT2) from the same organism, reveals how the identical ECF module adjusts to interact with the different substrate binding proteins FoIT2 and CbrT. ECF-CbrT is unrelated to the well-characterized B12 transporter BtuCDF, but their biochemical features indicate functional convergence.

  • 27. Sauri, Ana
    et al.
    Soprova, Zora
    Wickström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Van der Schors, Roel C
    Smit, August B
    Jong, Wouter S P
    Luirink, Joen
    The Bam (Omp85) complex is involved in secretion of the autotransporter haemoglobin protease.2009In: Microbiology (Reading, England), ISSN 1465-2080, Vol. 155, no Pt 12, p. 3982-91Article in journal (Refereed)
    Abstract [en]

    Autotransporters are large virulence factors secreted by Gram-negative bacteria. They are synthesized with a C-terminal domain that forms a beta-barrel pore in the outer membrane implicated in translocation of the upstream 'passenger' domain across the outer membrane. However, recent structural data suggest that the diameter of the beta-barrel pore is not sufficient to allow the passage of partly folded structures observed for several autotransporters. Here, we have used a stalled translocation intermediate of the autotransporter Hbp to identify components involved in insertion and translocation of the protein across the outer membrane. At this intermediate stage the beta-domain was not inserted and folded as an integral beta-barrel in the outer membrane whereas part of the passenger was surface exposed. The intermediate was copurified with the periplasmic chaperone SurA and subunits of the Bam (Omp85) complex that catalyse the insertion and assembly of outer-membrane proteins. The data suggest a critical role for this general machinery in the translocation of autotransporters across the outer membrane.

  • 28.
    Schlegel, Susan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Genevaux, Pierre
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    De-convoluting the Genetic Adaptations of E-coli C41(DE3) in Real Time Reveals How Alleviating Protein Production Stress Improves Yields2015In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 10, no 10, p. 1758-1766Article in journal (Refereed)
    Abstract [en]

    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.

  • 29. Schlegel, Susan
    et al.
    Genevaux, Pierre
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Isolating Escherichia coli strains for recombinant protein production2017In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 74, no 5, p. 891-908Article, review/survey (Refereed)
    Abstract [en]

    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.

  • 30.
    Schlegel, Susan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hjelm, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Baumgarten, Thomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Vikström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bacterial-based membrane protein production2014In: Biochimica et Biophysica Acta. Molecular Cell Research, ISSN 0167-4889, E-ISSN 1879-2596, Vol. 1843, no 8, p. 1739-1749Article, review/survey (Refereed)
    Abstract [en]

    Escherichia coli is by far the most widely used bacterial host for the production of membrane proteins. Usually, different strains, culture conditions and production regimes are screened for to design the optimal production process. However, these E. coli-based screening approaches often do not result in satisfactory membrane protein production yields. Recently, it has been shown that (i) E. coli strains with strongly improved membrane protein production characteristics can be engineered or selected for, (ii) many membrane proteins can be efficiently produced in E. coli-based cell-free systems, (iii) bacteria other than E. coli can be used for the efficient production of membrane proteins, and, (iv) membrane protein variants that retain functionality but are produced at higher yields than the wild-type protein can be engineered or selected for. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

  • 31.
    Schlegel, Susan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Klepsch, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Gialama, Dimitra
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wickström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Slotboom, Dirk Jan
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Revolutionizing membrane protein overexpression in bacteria2010In: Microbial biotechnology, ISSN 1751-7915, Vol. 3, no 4, p. 403-11Article in journal (Refereed)
    Abstract [en]

    The bacterium Escherichia coli is the most widely used expression host for overexpression trials of membrane proteins. Usually, different strains, culture conditions and expression regimes are screened for to identify the optimal overexpression strategy. However, yields are often not satisfactory, especially for eukaryotic membrane proteins. This has initiated a revolution of membrane protein overexpression in bacteria. Recent studies have shown that it is feasible to (i) engineer or select for E. coli strains with strongly improved membrane protein overexpression characteristics, (ii) use bacteria other than E. coli for the expression of membrane proteins, (iii) engineer or select for membrane protein variants that retain functionality but express better than the wild-type protein, and (iv) express membrane proteins using E. coli-based cell-free systems.

  • 32.
    Schlegel, Susan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Klepsch, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wickström, David
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wagner, Samuel
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Comparative analysis of cytoplasmic membrane proteomes of Escherichia coli using 2D blue native/SDS-PAGE2010In: Vol. 619, p. 257-69Article in journal (Refereed)
    Abstract [en]

    Two-dimensional blue native (2D BN)/SDS-PAGE is the method of choice for the global analysis of the subunits of complexes in membrane proteomes. In the 1st dimension complexes are separated by BN-PAGE, and in the 2nd dimension their subunits are resolved by SDS-PAGE. The currently available protocols result in the distortion of the 1st dimension BN-gel lanes during their transfer to the 2nd dimension separation gels. This leads to low reproducibility and high variation of 2D BN/SDS-gels, making 2D BN/SDS-PAGE unsuitable for comparative analysis. Here, we present a 2D BN/SDS-PAGE protocol where the 1st dimension BN-gel is cast on a GelBond PAG film. Immobilization prevents distortion of BN-gel lanes when they are transferred to the 2nd dimension, which lowers variation and greatly improves reproducibility of 2D BN/SDS-gels. The use of 2D BN/SDS-PAGE with an immobilized first dimension is illustrated by the characterization of the cytoplasmic membrane proteome of Escherichia coli cells overexpressing cytochrome bo (3).

  • 33.
    Schlegel, Susan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Löfblom, John
    Lee, Chiara
    Hjelm, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Klepsch, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Strous, Marc
    Drew, David
    Slotboom, Dirk Jan
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Optimizing Membrane Protein Overexpression in the Escherichia coli strain Lemo21(DE3)2012In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 423, no 4, p. 648-659Article in journal (Refereed)
    Abstract [en]

    Escherichia coli BL21(DE3) is widely used to overexpress proteins. In this overexpression host, the gene encoding the target protein is located on a plasmid and is under control of the T7 promoter, which is recognized exclusively by the T7 RNA polymerase (RNAP). The 17 RNAP gene is localized on the chromosome, and its expression is governed by the non-titratable, IPTG-inducible lacUV5 promoter. Recently, we constructed the Lemo21(DE3) strain, which allows improved control over the expression of genes from the 17 promoter. Lemo21(DE3) is a BL21(DE3) strain equipped with a plasmid harboring the gene encoding T7 lysozyme, an inhibitor of the T7 RNAP, under control of the exceptionally well-titratable rhamnose promoter. The overexpression yields of a large collection of membrane proteins in Lemo21(DE3) at different concentrations of rhamnose indicated that this strain may be very suitable for optimizing the production of membrane proteins. However, insight in the mechanism by which optimized expression yields are achieved in Lemo21(DE3) is lacking. Furthermore, whether the overexpressed proteins are suitable for functional and structural studies remains to be tested. Here, we show that in Lemo21(DE3), (i) the modulation of the activity of the 17 RNAP by the 17 lysozyme is key to optimizing the ratio of membrane proteins properly inserted in the cytoplasmic membrane to non-inserted proteins; (ii) maximizing the yields of membrane proteins is accompanied by reduction of the adverse effects of membrane protein overexpression, resulting in stable overexpression; and (iii) produced membrane proteins can be used for functional and structural studies.

  • 34.
    Schlegel, Susan
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Rujas, Edurne
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ytterberg, Anders Jimmy
    Zubarev, Roman A.
    Luirink, Joen
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Optimizing heterologous protein production in the periplasm of E. coli by regulating gene expression levels2013In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 12, p. 24-Article in journal (Refereed)
    Abstract [en]

    Background: In Escherichia coli many heterologous proteins are produced in the periplasm. To direct these proteins to the periplasm, they are equipped with an N-terminal signal sequence so that they can traverse the cytoplasmic membrane via the protein-conducting Sec-translocon. For poorly understood reasons, the production of heterologous secretory proteins is often toxic to the cell thereby limiting yields. To gain insight into the mechanism(s) that underlie this toxicity we produced two secretory heterologous proteins, super folder green fluorescent protein and a single-chain variable antibody fragment, in the Lemo21(DE3) strain. In this strain, the expression intensity of the gene encoding the target protein can be precisely controlled. Results: Both SFGFP and the single-chain variable antibody fragment were equipped with a DsbA-derived signal sequence. Producing these proteins following different gene expression levels in Lemo21(DE3) allowed us to identify the optimal expression level for each target gene. Too high gene expression levels resulted in saturation of the Sec-translocon capacity as shown by hampered translocation of endogenous secretory proteins and a protein misfolding/aggregation problem in the cytoplasm. At the optimal gene expression levels, the negative effects of the production of the heterologous secretory proteins were minimized and yields in the periplasm were optimized. Conclusions: Saturating the Sec-translocon capacity can be a major bottleneck hampering heterologous protein production in the periplasm. This bottleneck can be alleviated by harmonizing expression levels of the genes encoding the heterologous secretory proteins with the Sec-translocon capacity. Mechanistic insight into the production of proteins in the periplasm is key to optimizing yields in this compartment.

  • 35.
    Wagner, Samuel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Klepsch, Mirjam M.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Schlegel, Susan
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Appel, Ansgar
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Draheim, Roger
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tarry, Michael
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Högbom, Martin
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    van Wijk, Klaas J.
    Slotboom, Dirk J.
    Persson, Jan O.
    Stockholm University, Faculty of Science, Department of Mathematics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Tuning Escherichia coli for membrane protein overexpression2008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 38, p. 14371-17376Article in journal (Refereed)
    Abstract [en]

    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.

  • 36.
    Wagner, Samuel
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Pop, Ovidiu
    Haan, Gert-Jan
    Baars, Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Klepsch, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Genevaux, Pierre
    Luirink, Joen
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Biogenesis of MalF and the MalFGK2 maltose transport complex in Escherichia coli requires YidC2008In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, no 283, p. 17881-17890Article in journal (Refereed)
    Abstract [en]

    The polytopic inner membrane protein MalF is a constituent of the MalFGK2 maltose transport complex in Escherichia coli. We have studied the biogenesis of MalF using a combination of in vivo and in vitro approaches. MalF is targeted via the SRP pathway to the Sec/YidC insertion site. Despite close proximity of nascent MalF to YidC during insertion, YidC is not required for the insertion of MalF into the membrane. However, YidC is required for the stability of MalF and the formation of the MalFGK2 maltose transport complex. Our data indicate that YidC supports the folding of MalF into a stable conformation before it is incorporated into the maltose transport complex.

  • 37. Wickstrom, David
    et al.
    Wagner, Samuel
    Baars, Louise
    Ytterberg, A. Jimmy
    Klepsch, Mirjam
    van Wijk, Klaas J.
    Luirink, Joen
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Consequences of Depletion of the Signal Recognition Particle in Escherichia coli2011In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 286, no 6, p. 4598-4609Article in journal (Refereed)
    Abstract [en]

    Thus far, the role of the Escherichia coli signal recognition particle (SRP) has only been studied using targeted approaches. It has been shown for a handful of cytoplasmic membrane proteins that their insertion into the cytoplasmic membrane is at least partially SRP-dependent. Furthermore, it has been proposed that the SRP plays a role in preventing toxic accumulation of mistargeted cytoplasmic membrane proteins in the cytoplasm. To complement the targeted studies on SRP, we have studied the consequences of the depletion of the SRP component Fifty-four homologue (Ffh) in E. coli using a global approach. The steady-state proteomes and the proteome dynamics were evaluated using one- and two-dimensional gel analysis, followed by mass spectrometry-based protein identification and immunoblotting. Our analysis showed that depletion of Ffh led to the following: (i) impaired kinetics of the biogenesis of the cytoplasmic membrane proteome; (ii) lowered steady-state levels of the respiratory complexes NADH dehydrogenase, succinate dehydrogenase, and cytochrome bo(3) oxidase and lowered oxygen consumption rates; (iii) increased levels of the chaperones DnaK and GroEL at the cytoplasmic membrane; (iv) a sigma(32) stress response and protein aggregation in the cytoplasm; and (v) impaired protein synthesis. Our study shows that in E. coli SRP-mediated protein targeting is directly linked to maintaining protein homeostasis and the general fitness of the cell.

  • 38.
    Wickström, David
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Wagner, Samuel
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Simonsson, Per
    Pop, Ovidiu
    Baars, Louise
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ytterberg, A. Jimmy
    van Wijk, Klaas J.
    Luirink, Joen
    de Gier, Jan-Willem L.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Characterization of the Consequences of YidC Depletion on the Inner Membrane Proteome of E. coli Using 2D Blue Native/SDS-PAGE2011In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 409, no 2, p. 124-135Article in journal (Refereed)
    Abstract [en]

    In the bacterium Escherichia coli, the essential inner membrane protein (IMP) YidC assists in the biogenesis of IMPs and IMP complexes. Our current ideas about the function of YidC are based on targeted approaches using only a handful of model IMPs. Proteome-wide approaches are required to further our understanding of the significance of YidC and to find new YidC substrates. Here, using two-dimensional blue native/SDS-PAGE methodology that is suitable for comparative analysis, we have characterized the consequences of YidC depletion for the steady-state levels and oligomeric state of the constituents of the inner membrane proteome. Our analysis showed that (i) YidC depletion reduces the levels of a variety of complexes without changing their composition, (ii) the levels of IMPs containing only soluble domains smaller than 100 amino acids are likely to be reduced upon YidC depletion, whereas the levels of IMPs with at least one soluble domain larger than 100 amino acids do not, and (iii) the levels of a number of proteins with established or putative chaperone activity (HflC, HflK, PpiD, OppA, GroEL and DnaK) are strongly increased in the inner membrane fraction upon YidC depletion. In the absence of YidC, these proteins may assist the folding of sizeable soluble domains of IMPs, thereby supporting their folding and oligomeric assembly. In conclusion, our analysis identifies many new IMPs/IMP complexes that depend on YidC for their biogenesis, responses that accompany depletion of YidC and an IMP characteristic that is associated with YidC dependence.

  • 39. Yu, Zhong
    et al.
    Laven, Marielle
    Klepsch, Mirjam
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Bitter, Wilbert
    van Ulsen, Peter
    Luirink, Joen
    Role for Escherichia coli YidD in Membrane Protein Insertion2011In: Journal of Bacteriology, ISSN 0021-9193, E-ISSN 1098-5530, Vol. 193, no 19, p. 5242-5251Article in journal (Refereed)
    Abstract [en]

    YidC has an essential but poorly defined function in membrane protein insertion and folding in bacteria. The yidC gene is located in a gene cluster that is highly conserved in Gram-negative bacteria, the gene order being rpmH, rnpA, yidD, yidC, and trmE. Here, we show that Escherichia coli yidD, which overlaps with rnpA and is only 2 bp upstream of yidC, is expressed and localizes to the inner membrane, probably through an amphipathic helix. Inactivation of yidD had no discernible effect on cell growth and viability. However, compared to control cells, Delta yidD cells were affected in the insertion and processing of three YidC-dependent inner membrane proteins. Furthermore, in vitro cross-linking showed that YidD is in proximity of a nascent inner membrane protein during its localization in the Sec-YidC translocon, suggesting that YidD might be involved in the insertion process.

  • 40.
    Zhang, Zhe
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Ampah-Korsah, Henry
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Karyolaimos, Alexandros
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Construction and characterization of the bacteriophage testable BL21(DE3)-derivative BL21T7Manuscript (preprint) (Other academic)
  • 41.
    Zhang, Zhe
    et al.
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Kuipers, Grietje
    Niemiec, Lukasz
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Baumgarten, Thomas
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Slotboom, Dirk Jan
    de Gier, Jan-Willem
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    Hjelm, Anna
    Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
    High-level production of membrane proteins in E-coli BL21(DE3) by omitting the inducer IPTG2015In: Microbial Cell Factories, ISSN 1475-2859, E-ISSN 1475-2859, Vol. 14, article id 142Article in journal (Refereed)
    Abstract [en]

    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-beta-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.

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