Protein Expression and Purification Core FacilityPublications
Use of the Uteroglobin Platform for the Expression of a Bivalent Antibody against Oncofetal Fibronectin in Escherichia coli.
Ventura, E., Riondato, M., Sambuceti, G., Salis, A., Damonte, G., Cordazzo, C., Besir, H., Pistoia, V. & Zardi, L.
PLoS One. 2013 Dec 19;8(12):e82878. doi: 10.1371/journal.pone.0082878.
Escherichia coli is a robust, economic and rapid expression system for the production of recombinant therapeutic proteins. However, the expression in bacterial systems of complex molecules such as antibodies and fusion proteins is still affected by several drawbacks. We have previously described a procedure based on uteroglobin (UG) for the engineering of very soluble and stable polyvalent and polyspecific fusion proteins in mammalian cells (Ventura et al. 2009. J. Biol. Chem. 284ratio26646-26654.) Here, we applied the UG platform to achieve the expression in E. coli of a bivalent human recombinant antibody (L19) toward the oncofetal fibronectin (B-FN), a pan-tumor target. Purified bacterial L19-UG was highly soluble, stable, and, in all molecules, the L19 moiety maintained its immunoreactivity. About 50-70% of the molecules were covalent homodimer, however after refolding with the redox couple reduced-glutathione/oxidized-glutathione (GSH/GSSG), 100% of molecules were covalent dimers. Mass spectrometry studies showed that the proteins produced by E. coli and mammalian cells have an identical molecular mass and that both proteins are not glycosylated. L19-UG from bacteria can be freeze-dried without any loss of protein and immunoreactivity. In vivo, in tumor-bearing mice, radio-iodinated L19-UG selectively accumulated in neoplastic tissues showing the same performance of L19-UG from mammalian cells. The UG-platform may represent a general procedure for production of various biological therapeutics in E. coli.
The novel Fh8 and H fusion partners for soluble protein expression in Escherichia coli: a comparison with the traditional gene fusion technology.
Costa, S.J., Almeida, A., Castro, A., Domingues, L. & Besir, H.
Appl Microbiol Biotechnol. 2013 Aug;97(15):6779-91. doi:10.1007/s00253-012-4559-1. Epub 2012 Nov 20.
The Escherichia coli host system is an advantageous choice for simple and inexpensive recombinant protein production but it still presents bottlenecks at expressing soluble proteins from other organisms. Several efforts have been taken to overcome E. coli limitations, including the use of fusion partners that improve protein expression and solubility. New fusion technologies are emerging to complement the traditional solutions. This work evaluates two novel fusion partners, the Fh8 tag (8 kDa) and the H tag (1 kDa), as solubility enhancing tags in E. coli and their comparison to commonly used fusion partners. A broad range comparison was conducted in a small-scale screening and subsequently scaled-up. Six difficult-to-express target proteins (RVS167, SPO14, YPK1, YPK2, Frutalin and CP12) were fused to eight fusion tags (His, Trx, GST, MBP, NusA, SUMO, H and Fh8). The resulting protein expression and solubility levels were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis before and after protein purification and after tag removal. The Fh8 partner improved protein expression and solubility as the well-known Trx, NusA or MBP fusion partners. The H partner did not function as a solubility tag. Cleaved proteins from Fh8 fusions were soluble and obtained in similar or higher amounts than proteins from the cleavage of other partners as Trx, NusA or MBP. The Fh8 fusion tag therefore acts as an effective solubility enhancer, and its low molecular weight potentially gives it an advantage over larger solubility tags by offering a more reliable assessment of the target protein solubility when expressed as a fusion protein.
A new method to customize protein expression vectors for fast, efficient and background free parallel cloning.
Scholz, J., Besir, H., Strasser, C. & Suppmann, S.
BMC Biotechnol. 2013 Feb 14;13:12. doi: 10.1186/1472-6750-13-12.
BACKGROUND: Expression and purification of correctly folded proteins typically require screening of different parameters such as protein variants, solubility enhancing tags or expression hosts. Parallel vector series that cover all variations are available, but not without compromise. We have established a fast, efficient and absolutely background free cloning approach that can be applied to any selected vector. RESULTS: Here we describe a method to tailor selected expression vectors for parallel Sequence and Ligation Independent Cloning. SLIC cloning enables precise and sequence independent engineering and is based on joining vector and insert with 15-25 bp homologies on both DNA ends by homologous recombination. We modified expression vectors based on pET, pFastBac and pTT backbones for parallel PCR-based cloning and screening in E.coli, insect cells and HEK293E cells, respectively. We introduced the toxic ccdB gene under control of a strong constitutive promoter for counterselection of insert less vector. In contrast to DpnI treatment commonly used to reduce vector background, ccdB used in our vector series is 100% efficient in killing parental vector carrying cells and reduces vector background to zero. In addition, the 3' end of ccdB functions as a primer binding site common to all vectors. The second shared primer binding site is provided by a HRV 3C protease cleavage site located downstream of purification and solubility enhancing tags for tag removal. We have so far generated more than 30 different parallel expression vectors, and successfully cloned and expressed more than 250 genes with this vector series. There is no size restriction for gene insertion, clone efficiency is > 95% with clone numbers up to 200. The procedure is simple, fast, efficient and cost-effective. All expression vectors showed efficient expression of eGFP and different target proteins requested to be produced and purified at our Core Facility services. CONCLUSION: This new expression vector series allows efficient and cost-effective parallel cloning and thus screening of different protein constructs, tags and expression hosts.
The protein interaction network of a taxis signal transduction system in a Halophilic Archaeon.
Schlesner, M., Miller, A., Besir, H., Aivaliotis, M., Streif, J., Scheffer, B., Siedler, F. & Oesterhelt, D.
BMC Microbiol. 2012 Nov 21;12:272. doi: 10.1186/1471-2180-12-272.
ABSTRACT: BACKGROUND: The taxis signaling system of the extreme halophilic archaeon Halobacterium (Hbt.) salinarum differs in several aspects from its model bacterial counterparts Escherichia coli and Bacillus subtilis. We studied the protein interactions in the Hbt. salinarum taxis signaling system to gain an understanding of its structure, to gain knowledge about its known components and to search for new members. RESULTS: The interaction analysis revealed that the core signaling proteins are involved in different protein complexes and our data provide evidence for dynamic interchanges between them. Fifteen of the eighteen taxis receptors (halobacterial transducers, Htrs) can be assigned to four different groups depending on their interactions with the core signaling proteins. Only one of these groups, which contains six of the eight Htrs with known signals, shows the composition expected for signaling complexes (receptor, kinase CheA, adaptor CheW, response regulator CheY). From the two Hbt. salinarum CheW proteins, only CheW1 is engaged in signaling complexes with Htrs and CheA, whereas CheW2 interacts with Htrs but not with CheA. CheY connects the core signaling structure to a subnetwork consisting of the two CheF proteins (which build a link to the flagellar apparatus), CheD (the hub of the subnetwork), two CheC complexes and the receptor methylesterase CheB. CONCLUSIONS: Based on our findings, we propose two hypotheses. First, Hbt. salinarum might have the capability to dynamically adjust the impact of certain Htrs or Htr clusters depending on its current needs or environmental conditions. Secondly, we propose a hypothetical feedback loop from the response regulator to Htr methylation made from the CheC proteins, CheD and CheB, which might contribute to adaptation analogous to the CheC/CheD system of B. subtilis.
GAGE Cancer-Germline Antigens Are Recruited to the Nuclear Envelope by Germ Cell-Less (GCL).
Gjerstorff, M.F., Rosner, H.I., Pedersen, C.B., Greve, K.B., Schmidt, S., Wilson, K.L., Mollenhauer, J., Besir, H., Poulsen, F.M., Mollegaard, N.E. & Ditzel, H.J.
PLoS One. 2012;7(9):e45819. doi: 10.1371/journal.pone.0045819. Epub 2012 Sep 20.
GAGE proteins are highly similar, primate-specific molecules with unique primary structure and undefined cellular roles. They are restricted to cells of the germ line in adult healthy individuals, but are broadly expressed in a wide range of cancers. In a yeast two-hybrid screen we identified the metazoan transcriptional regulator, Germ cell-less (GCL), as an interaction partner of GAGE12I. GCL directly binds LEM-domain proteins (LAP2beta, emerin, MAN1) at the nuclear envelope, and we found that GAGE proteins were recruited to the nuclear envelope inner membrane by GCL. Based on yeast two-hybrid analysis and pull-down experiments of GCL polypeptides, GCL residues 209-320 (which includes the BACK domain) were deduced sufficient for association with GAGE proteins. GAGE mRNAs and GCL mRNA were demonstrated in human testis and most types of cancers, and at the protein level GAGE members and GCL were co-expressed in cancer cell lines. Structural studies of GAGE proteins revealed no distinct secondary or tertiary structure, suggesting they are intrinsically disordered. Interestingly GAGE proteins formed stable complexes with dsDNA in vitro at physiological concentrations, and GAGE12I bound several different dsDNA fragments, suggesting sequence-nonspecific binding. Dual association of GAGE family members with GCL at the nuclear envelope inner membrane in cells, and with dsDNA in vitro, implicate GAGE proteins in chromatin regulation in germ cells and cancer cells.
Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice.
Bayer-Giraldi, M., Weikusat, I., Besir, H. & Dieckmann, G.
Cryobiology. 2011 Dec;63(3):210-9. Epub 2011 Aug 26.
Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9
Expression, purification and characterization of the cancer-germline antigen GAGE12I: a candidate for cancer immunotherapy.
Gjerstorff, M.F., Besir, H., Larsen, M.R. & Ditzel, H.J.
Protein Expr Purif. 2010 Oct;73(2):217-22. Epub 2010 May 27.
GAGE cancer-germline antigens are frequently expressed in a broad range of different cancers, while their expression in normal tissues is limited to the germ cells of the immune privileged organs, testis and ovary. GAGE proteins are immunogenic in humans, which make them promising targets for immunotherapy and candidates for cancer vaccines. Recombinant proteins may be superior to peptides as immunogens, since they have the potential to prime both CD4(+) and CD8(+) T cells and are not dependent on patient HLA-type. We have developed a method for production of highly pure recombinant GAGE12I-His by intracellular expression in yeast (Pichia pastoris) and nickel affinity, ion exchange and gel filtration purification. The identity of the purified protein was confirmed by mass spectrometry. This strategy yielded a total of 48 mg of highly pure (>98%) GAGE12I from 8 L of culture (6 mg/l). Interestingly, gel filtration and formaldehyde cross-linking indicated that GAGE12I forms tetramers. The purified recombinant GAGE12I represents a candidate molecule for vaccination of cancer patients and will form the basis for further structural analysis of GAGE proteins.
Staining of proteins in gels with Coomassie G-250 without organic solvent and acetic acid.
Lawrence, A.M. & Besir, H.U.
J Vis Exp. 2009 Aug 14;(30). pii: 1350. doi: 10.3791/1350.
In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80 mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staining of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compounds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.
Absolute SILAC for accurate quantitation of proteins in complex mixtures down to the attomole level.
Hanke, S., Besir, H., Oesterhelt, D. & Mann, M.
J Proteome Res. 2008 Mar;7(3):1118-30. Epub 2008 Feb 14.
Mass spectrometry based proteomics can routinely identify hundreds of proteins in a single LC-MS run, and methods have been developed for relative quantitation between differentially treated samples using stable isotopes. However, absolute quantitation has so far required addition of a labeled standard late in the experimental workflow, introducing variability due to sample preparation. Here we present a new variant of the stable isotope labeling by amino acids in cell culture (SILAC) technique termed "Absolute SILAC" that allows accurate quantitation of selected proteins in complex mixtures. SILAC-labeled recombinant proteins produced in vivo or in vitro are used as internal standards, which are directly mixed into lysates of cells or tissues. This minimizes differences in sample processing between the isotope-labeled standard and its endogenous counterpart. We show that it is possible to quantify over several orders of magnitude, even in the background of a whole cell lysate. We furthermore devise a strategy to quantify peptides at or below their signal-to-noise level on hybrid ion trap instruments, shown here for the LTQ-Orbitrap. The data system triggers on peptides of the SILAC-labeled protein, initiating ion collection in a narrow mass range including the endogenous and labeled peptide. This strategy extends the regular detection limit of an LTQ-Orbitrap by at least an order of magnitude and accurately quantifies down to 150 attomole of protein in a cell lysate without any fractionation prior to LC-MS. We use Absolute SILAC to determine the copy number per cell of growth factor receptor-bound protein 2 (Grb2) in HeLa, HepG2, and C2C12 cells to 5.5 x 10(5), 8.8 x 10(5), and 5.7 x 10(5), respectively, in the exponential growth phase.
A small protein from the bop-brp intergenic region of Halobacterium salinarum contains a zinc finger motif and regulates bop and crtB1 transcription.
Tarasov, V.Y., Besir, H., Schwaiger, R., Klee, K., Furtwangler, K., Pfeiffer, F. & Oesterhelt, D.
Mol Microbiol. 2008 Feb;67(4):772-80. Epub 2008 Jan 2.
Bacteriorhodopsin, the photosynthetic protein of Halobacterium salinarum, is optimally expressed under anaerobic growth conditions. We identified Brz (OE3104F, bacteriorhodopsin-regulating zinc finger protein), a new regulator of the bop gene. It is a small protein with a zinc finger motif, encoded directly upstream of the bop gene in the same orientation. Deletion of the brz gene caused a large decrease of bop mRNA levels as shown by Northern blot and microarray analysis. A similar effect was obtained by site-directed mutagenesis of Cys and His residues in the zinc finger motif, indicating the importance of this motif for the function of the protein. In silico analysis of the genomes from H. salinarum and other archaea revealed a large family of similar small zinc finger motif proteins, some of which may also be involved in transcription regulation of their adjacent genes.
The low molecular weight proteome of Halobacterium salinarum.
Klein, C., Aivaliotis, M., Olsen, J.V., Falb, M., Besir, H., Scheffer, B., Bisle, B., Tebbe, A., Konstantinidis, K., Siedler, F., Pfeiffer, F., Mann, M. & Oesterhelt, D.
J Proteome Res. 2007 Apr;6(4):1510-8. Epub 2007 Feb 28.
Systematic investigation of low molecular weight proteins (LMW, below 20 kDa) in the archaeon Halobacterium salinarum resulted in a 6-fold enhancement of the identification rate, reaching 35% of the theoretical proteome in that size range. This was achieved by optimization of common protocols for protein analysis with general applicability. LMW proteins were rapidly and effectively enriched by filter membrane centrifugation followed by tricine SDS-PAGE. Without staining and with significantly shortened digestion protocols, LMW proteins were identified using an FT-ICR mass spectrometer which allows reliable protein identification by MS3 of a single peptide. In addition to a series of technical challenges, small proteins may show low gene expression levels as suggested by their low average codon adaptation index. Twenty functionally uncharacterized proteins contain a characteristic DNA/RNA binding zinc finger motif which underlines the biological relevance of the small proteome and the necessity of their analysis for systems biology.
Characterizing molecular interactions in different bacteriorhodopsin assemblies by single-molecule force spectroscopy.
Sapra, K.T., Besir, H., Oesterhelt, D. & Muller, D.J.
J Mol Biol. 2006 Jan 27;355(4):640-50. Epub 2005 Nov 17.
Using single-molecule force spectroscopy we characterized inter- and intramolecular interactions stabilizing structural segments of individual bacteriorhodopsin (BR) molecules assembled into trimers and dimers, and monomers. While the assembly of BR did not vary the location of these structural segments, their intrinsic stability could change up to 70% increasing from monomer to dimer to trimer. Since each stable structural segment established one unfolding barrier, we conclude that the locations of unfolding barriers were determined by intramolecular interactions but that their strengths were strongly influenced by intermolecular interactions. Subtracting the unfolding forces of the BR trimer from that of monomer allowed us to calculate the contribution of inter- and intramolecular interactions to the membrane protein stabilization. Statistical analyses showed that the unfolding pathways of differently assembled BR molecules did not differ in their appearance but in their population. This suggests that in our experiments the membrane protein assembly does not necessarily change the location of unfolding barriers within the protein, but certainly their strengths, and thus alters the probability of a protein to choose certain unfolding pathways.
Structure of a halophilic nucleoside diphosphate kinase from Halobacterium salinarum.
Besir, H., Zeth, K., Bracher, A., Heider, U., Ishibashi, M., Tokunaga, M. & Oesterhelt, D.
FEBS Lett. 2005 Dec 5;579(29):6595-600. Epub 2005 Nov 9.
Nucleoside diphosphate kinase from the halophilic archaeon Halobacterium salinarum was crystallized in a free state and a substrate-bound form with CDP. The structures were solved to a resolution of 2.35 and 2.2A, respectively. Crystals with the apo-form were obtained with His6-tagged enzyme, whereas the untagged form was used for co-crystallization with the nucleotide. Crosslinking under different salt and pH conditions revealed a stronger oligomerization tendency for the tagged protein at low and high salt concentrations. The influence of the His6-tag on the halophilic nature of the enzyme is discussed on the basis of the observed structural properties.
Membrane lateral mobility obstructed by polymer-tethered lipids studied at the single molecule level.
Deverall, M.A., Gindl, E., Sinner, E.K., Besir, H., Ruehe, J., Saxton, M.J. & Naumann, C.A.
Biophys J. 2005 Mar;88(3):1875-86. Epub 2004 Dec 21.
Obstructed long-range lateral diffusion of phospholipids (TRITC-DHPE) and membrane proteins (bacteriorhodopsin) in a planar polymer-tethered 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayer is studied using wide-field single molecule fluorescence microscopy. The obstacles are well-controlled concentrations of hydrophobic lipid-mimicking dioctadecylamine moieties in the polymer-exposed monolayer of the model membrane. Diffusion of both types of tracer molecules is well described by a percolating system with different percolation thresholds for lipids and proteins. Data analysis using a free area model of obstructed lipid diffusion indicates that phospholipids and tethered lipids interact via hard-core repulsion. A comparison to Monte Carlo lattice calculations reveals that tethered lipids act as immobile obstacles, are randomly distributed, and do not self-assemble into large-scale aggregates for low to moderate tethering concentrations. A procedure is presented to identify anomalous subdiffusion from tracking data at a single time lag. From the analysis of the cumulative distribution function of the square displacements, it was found that TRITC-DHPE and W80i show normal diffusion at lower concentrations of tethered lipids and anomalous diffusion at higher ones. This study may help improve our understanding of how lipids and proteins in biomembranes may be obstructed by very small obstacles comprising only one or very few molecules.
Structure of the light-driven chloride pump halorhodopsin at 1.8 A resolution.
Kolbe, M., Besir, H., Essen, L.O. & Oesterhelt, D.
Science. 2000 May 26;288(5470):1390-6.
Halorhodopsin, an archaeal rhodopsin ubiquitous in Haloarchaea, uses light energy to pump chloride through biological membranes. Halorhodopsin crystals were grown in a cubic lipidic phase, which allowed the x-ray structure determination of this anion pump at 1.8 angstrom resolution. Halorhodopsin assembles to trimers around a central patch consisting of palmitic acid. Next to the protonated Schiff base between Lys(242) and the isomerizable retinal chromophore, a single chloride ion occupies the transport site. Energetic calculations on chloride binding reveal a combination of ion-ion and ion-dipole interactions for stabilizing the anion 18 angstroms below the membrane surface. Ion dragging across the protonated Schiff base explains why chloride and proton translocation modes are mechanistically equivalent in archaeal rhodopsins.