Constitutive expression of active microbial transglutaminase in Escherichia coli and comparative characterization to a known variant.

BACKGROUND: Microbial transglutaminase (mTG) is a robust enzyme catalyzing the formation of an isopeptide bond between glutamine and lysine residues. It has found use in food applications, pharmaceuticals, textiles, and biomedicine. Overexpression of soluble and active mTG in E. coli has been limited due to improper protein folding and requirement for proteolytic cleavage of the pro-domain. Furthermore, to integrate mTG more fully industrially and academically, thermostable and solvent-stable variants may be imperative. RESULTS: A novel expression system constitutively producing active mTG was designed. Wild-type (WT) mTG and a S2P variant had similar expression levels, comparable to previous studies. Kinetic constants were determined by a glutamate dehydrogenase-coupled assay, and the S2P variant showed an increased affinity and a doubled enzyme efficiency towards Z-Gln-Gly. The melting temperature (Tm) of the WT was determined by intrinsic fluorescence measurements to be 55.8 ± 0.1 °C and of the S2P variant to be 56.3 ± 0.4 °C and 45.5 ± 0.1 °C, showing a moderately different thermostability profile. Stability in water miscible organic solvents was determined for both the WT and S2P variant. Of the solvents tested, incubation of mTG in isopropanol for 24 h at 4 °C showed the strongest stabilizing effect with mTG retaining 61 and 72% activity for WT and S2P respectively in 70% isopropanol. Both enzymes also showed an increased initial activity in the presence of organic solvents with the highest activity increase in 40% DMSO. Nevertheless, both enzymes were inactivated in 70% of all organic solvents tested. CONCLUSIONS: A constitutive expression system of active mTG in E. coli without downstream proteolytic cleavage processing was used for overexpression and characterization. High throughput techniques for testing thermostability and kinetics were useful in streamlining analysis and could be used in the future for quickly identifying beneficial mutants. Hitherto untested thermostability and stability of mTG in organic solvents was evaluated, which can pave the way for use of the enzyme in novel applications and processes.

Creating an Efficient Methanol-Stable Biocatalyst by Protein and Immobilization Engineering Steps towards Efficient Biosynthesis of Biodiesel.

Two ternary sol-gel matrices, an octyltriethoxysilane-based aliphatic matrix and a phenyltriethoxysilane (PTEOS)-based aromatic matrix, were used to immobilize a methanol-stable variant of lipase from Geobacillus stearothermophilus T6 for the synthesis of biodiesel from waste oil. Superior thermal stability of the mutant versus the wildtype in methanol was confirmed by intrinsic protein fluorescence measurements. The influence of skim milk and soluble E. coli lysate proteins as bulking and stabilizing agents in conjunction with sol-gel entrapment were investigated. E. coli lysate proteins were better stabilizing agents of the purified lipase mutant than skim milk, as evidenced by reverse engineering of the aromatic-based system. This was also shown for commercial Candida antarctica lipase B (CaLB) and Thermomyces lanuginosus lipase (TLL). Uniform, dense, and nonaggregated particles imaged by scanning electron microscopy and a small particle size of 13 µm pertaining to the system comprising PTEOS and E. coli lysate proteins correlated well with high esterification activity. Combining protein and immobilization engineering resulted in a durable biocatalyst with efficient recycling ability and high biodiesel conversion rates.

Structure-based in silico identification of ubiquitin-binding domains provides insights into the ALIX-V:ubiquitin complex and retrovirus budding.

The ubiquitylation signal promotes trafficking of endogenous and retroviral transmembrane proteins. The signal is decoded by a large set of ubiquitin (Ub) receptors that tether Ub-binding domains (UBDs) to the trafficking machinery. We developed a structure-based procedure to scan the protein data bank for hidden UBDs. The screen retrieved many of the known UBDs. Intriguingly, new potential UBDs were identified, including the ALIX-V domain. Pull-down, cross-linking and E3-independent ubiquitylation assays biochemically corroborated the in silico findings. Guided by the output model, we designed mutations at the postulated ALIX-V:Ub interface. Biophysical affinity measurements using microscale-thermophoresis of wild-type and mutant proteins revealed some of the interacting residues of the complex. ALIX-V binds mono-Ub with a K(d) of 119 µM. We show that ALIX-V oligomerizes with a Hill coefficient of 5.4 and IC(50) of 27.6 µM and that mono-Ub induces ALIX-V oligomerization. Moreover, we show that ALIX-V preferentially binds K63 di-Ub compared with mono-Ub and K48 di-Ub. Finally, an in vivo functionality assay demonstrates the significance of ALIX-V:Ub interaction in equine infectious anaemia virus budding. These results not only validate the new procedure, but also demonstrate that ALIX-V directly interacts with Ub in vivo and that this interaction can influence retroviral budding.

Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions.

Microscale thermophoresis (MST) allows for quantitative analysis of protein interactions in free solution and with low sample consumption. The technique is based on thermophoresis, the directed motion of molecules in temperature gradients. Thermophoresis is highly sensitive to all types of binding-induced changes of molecular properties, be it in size, charge, hydration shell or conformation. In an all-optical approach, an infrared laser is used for local heating, and molecule mobility in the temperature gradient is analyzed via fluorescence. In standard MST one binding partner is fluorescently labeled. However, MST can also be performed label-free by exploiting intrinsic protein UV-fluorescence. Despite the high molecular weight ratio, the interaction of small molecules and peptides with proteins is readily accessible by MST. Furthermore, MST assays are highly adaptable to fit to the diverse requirements of different biomolecules, such as membrane proteins to be stabilized in solution. The type of buffer and additives can be chosen freely. Measuring is even possible in complex bioliquids like cell lysate allowing close to in vivo conditions without sample purification. Binding modes that are quantifiable via MST include dimerization, cooperativity and competition. Thus, its flexibility in assay design qualifies MST for analysis of biomolecular interactions in complex experimental settings, which we herein demonstrate by addressing typically challenging types of binding events from various fields of life science.

Label-free microscale thermophoresis discriminates sites and affinity of protein-ligand binding.

Look, no label! Microscale thermophoresis makes use of the intrinsic fluorescence of proteins to quantify the binding affinities of ligands and discriminate between binding sites. This method is suitable for studying binding interactions of very small amounts of protein in solution. The binding of ligands to iGluR membrane receptors, small-molecule inhibitorss to kinase p38, aptamers to thrombin, and Ca(2+) ions to synaptotagmin was quantified.

Rational design of small molecule inhibitors targeting RhoA subfamily Rho GTPases.

Rho GTPases have been implicated in diverse cellular functions and are potential therapeutic targets. By virtual screening, we have identified a Rho-specific inhibitor, Rhosin. Rhosin contains two aromatic rings tethered by a linker, and it binds to the surface area sandwiching Trp58 of RhoA with a submicromolar Kd and effectively inhibits GEF-catalyzed RhoA activation. In cells, Rhosin specifically inhibited RhoA activity and RhoA-mediated cellular function without affecting Cdc42 or Rac1 signaling activities. By suppressing RhoA or RhoC activity, Rhosin could inhibit mammary sphere formation by breast cancer cells, suppress invasion of mammary epithelial cells, and induce neurite outgrowth of PC12 cells in synergy with NGF. Thus, the rational designed RhoA subfamily-specific small molecule inhibitor is useful for studying the physiological and pathologic roles of Rho GTPase.

Microscale thermophoresis as a sensitive method to quantify protein: nucleic acid interactions in solution.

Microscale thermophoresis (MST) is a new method that enables the quantitative analysis of molecular interactions in solution at the microliter scale. The technique is based on the thermophoresis of molecules, which provides information about molecule size, charge, and hydration shell. Since at least one of these parameters is typically affected upon binding, the method can be used for the analysis of each kind of biomolecular interaction or modification of proteins or DNA. Quantitative binding parameters are obtained by using a serial dilution of the binding substrate. This section provides a detailed protocol describing the analysis of DNA-protein interactions, using the AT-hook peptides as a model system that bind to short double-stranded DNA.

Designer lipid-like peptides: a class of detergents for studying functional olfactory receptors using commercial cell-free systems.

A crucial bottleneck in membrane protein studies, particularly G-protein coupled receptors, is the notorious difficulty of finding an optimal detergent that can solubilize them and maintain their stability and function. Here we report rapid production of 12 unique mammalian olfactory receptors using short designer lipid-like peptides as detergents. The peptides were able to solubilize and stabilize each receptor. Circular dichroism showed that the purified olfactory receptors had alpha-helical secondary structures. Microscale thermophoresis suggested that the receptors were functional and bound their odorants. Blot intensity measurements indicated that milligram quantities of each olfactory receptor could be produced with at least one peptide detergent. The peptide detergents' capability was comparable to that of the detergent Brij-35. The ability of 10 peptide detergents to functionally solubilize 12 olfactory receptors demonstrates their usefulness as a new class of detergents for olfactory receptors, and possibly other G-protein coupled receptors and membrane proteins.

A robust and rapid method of producing soluble, stable, and functional G-protein coupled receptors.

Membrane proteins, particularly G-protein coupled receptors (GPCRs), are notoriously difficult to express. Using commercial E. coli cell-free systems with the detergent Brij-35, we could rapidly produce milligram quantities of 13 unique GPCRs. Immunoaffinity purification yielded receptors at >90% purity. Secondary structure analysis using circular dichroism indicated that the purified receptors were properly folded. Microscale thermophoresis, a novel label-free and surface-free detection technique that uses thermal gradients, showed that these receptors bound their ligands. The secondary structure and ligand-binding results from cell-free produced proteins were comparable to those expressed and purified from HEK293 cells. Our study demonstrates that cell-free protein production using commercially available kits and optimal detergents is a robust technology that can be used to produce sufficient GPCRs for biochemical, structural, and functional analyses. This robust and simple method may further stimulate others to study the structure and function of membrane proteins.

Molecular interaction studies using microscale thermophoresis.

Abstract The use of infrared laser sources for creation of localized temperature fields has opened new possibilities for basic research and drug discovery. A recently developed technology, Microscale Thermophoresis (MST), uses this temperature field to perform biomolecular interaction studies. Thermophoresis, the motion of molecules in temperature fields, is very sensitive to changes in size, charge, and solvation shell of a molecule and thus suited for bioanalytics. This review focuses on the theoretical background of MST and gives a detailed overview on various applications to demonstrate the broad applicability. Experiments range from the quantification of the affinity of low-molecular-weight binders using fluorescently labeled proteins, to interactions between macromolecules and multi-component complexes like receptor containing liposomes. Information regarding experiment and experimental setup is based on the Monolith NT.115 instrument (NanoTemper Technologies GmbH).

Peptide surfactants for cell-free production of functional G protein-coupled receptors.

Two major bottlenecks in elucidating the structure and function of membrane proteins are the difficulty of producing large quantities of functional receptors, and stabilizing them for a sufficient period of time. Selecting the right surfactant is thus crucial. Here we report using peptide surfactants in commercial Escherichia coli cell-free systems to rapidly produce milligram quantities of soluble G protein-coupled receptors (GPCRs). These include the human formyl peptide receptor, human trace amine-associated receptor, and two olfactory receptors. The GPCRs expressed in the presence of the peptide surfactants were soluble and had α-helical secondary structures, suggesting that they were properly folded. Microscale thermophoresis measurements showed that one olfactory receptor expressed using peptide surfactants bound its known ligand heptanal (molecular weight 114.18). These short and simple peptide surfactants may be able to facilitate the rapid production of GPCRs, or even other membrane proteins, for structure and function studies.

The TGF-ß signaling modulators TRAP1/TGFBRAP1 and VPS39/Vam6/TLP are essential for early embryonic development.

The pleiotropic cytokine transforming growth factor-ß (TGF-ß) signals through different pathways among which the Smad- and the MAP-Kinase pathways are already well characterized. Both pathways utilize adaptor/chaperone molecules that facilitate or modulate the intracellular signaling events. Two of the proteins shown in vitro to play a role in Smad-dependent signaling are the TGF-ß Receptor Associated Protein-1 (TRAP1, also TGFBRAP1) and its homologue VPS39, also known as Vam6 and TRAP1-Like-Protein (TLP). We generated mice deficient for TRAP1 and VPS39/TLP, respectively. Absence of TRAP1 protein results in death at either of two defined timepoints during embryogenesis, before the blastula stage or during gastrulation, whereas most of the VPS39 deficient mice die before E6.5. Heterozygous mice show no overt phenotype. In summary, our data indicate that TRAP1 and VPS39 are nonredundant and essentially required for early embryonic development.

Reciprocal relationship between O6-methylguanine-DNA methyltransferase P140K expression level and chemoprotection of hematopoietic stem cells.

Retroviral-mediated delivery of the P140K mutant O(6)-methylguanine-DNA methyltransferase (MGMT(P140K)) into hematopoietic stem cells (HSC) has been proposed as a means to protect against dose-limiting myelosuppressive toxicity ensuing from chemotherapy combining O(6)-alkylating agents (e.g., temozolomide) with pseudosubstrate inhibitors (such as O(6)-benzylguanine) of endogenous MGMT. Because detoxification of O(6)-alkylguanine adducts by MGMT is stoichiometric, it has been suggested that higher levels of MGMT will afford better protection to gene-modified HSC. However, accomplishing this goal would potentially be in conflict with current efforts in the gene therapy field, which aim to incorporate weaker enhancer elements to avoid insertional mutagenesis. Using a panel of self-inactivating gamma-retroviral vectors that express a range of MGMT(P140K) activity, we show that MGMT(P140K) expression by weaker cellular promoter/enhancers is sufficient for in vivo protection/selection following treatment with O(6)-benzylguanine/temozolomide. Conversely, the highest level of MGMT(P140K) activity did not promote efficient in vivo protection despite mediating detoxification of O(6)-alkylguanine adducts. Moreover, very high expression of MGMT(P140K) was associated with a competitive repopulation defect in HSC. Mechanistically, we show a defect in cellular proliferation associated with elevated expression of MGMT(P140K), but not wild-type MGMT. This proliferation defect correlated with increased localization of MGMT(P140K) to the nucleus/chromatin. These data show that very high expression of MGMT(P140K) has a deleterious effect on cellular proliferation, engraftment, and chemoprotection. These studies have direct translational relevance to ongoing clinical gene therapy studies using MGMT(P140K), whereas the novel mechanistic findings are relevant to the basic understanding of DNA repair by MGMT.

Gene therapy of beta(c)-deficient pulmonary alveolar proteinosis (beta(c)-PAP): studies in a murine in vivo model.

Pulmonary alveolar proteinosis (PAP) due to deficiency of the common beta-chain (beta(c)) of the interleukin-3 (IL-3)/IL-5/granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors is a rare monogeneic disease characterized by functional insufficiency of pulmonary macrophages. Hematopoietic stem cell gene therapy for restoring expression of beta(c)-protein in the hematopoietic system may offer a curative approach. Toward this end, we generated a retroviral construct expressing the murine beta(c) (mbeta(c)) gene and conducted investigations in a murine model of beta(c)-deficient PAP. Functional correction of mbeta(c) activity in mbeta(c)(-/-) bone marrow (BM) cells was demonstrated by restoration of in vitro colony formation in response to GM-CSF. In addition, in a murine in vivo model of mbeta(c)-deficient PAP mbeta(c) gene transfer to hematopoietic stem cells not only restored the GM-CSF-sensitivity of hematopoietic progenitor cells but also, within a period of 12 weeks, almost completely reversed the morphologic features of surfactant accumulation. These results were obtained despite modest transduction levels (10-20%) and, in comparison to wild-type mice, clearly reduced beta(c) expression levels were detected in hematopoietic cells. Therefore, our data demonstrating genetic and functional correction of mbeta(c)(-/-) deficiency in vitro as well as in a murine in vivo model of PAP strongly suggest gene therapy as a potential new treatment modality in beta(c)-deficient PAP.

Gene Therapy of ßc-Deficient Pulmonary Alveolar Proteinosis (ßc-PAP): Studies in a Murine in vivo Model.

Pulmonary alveolar proteinosis (PAP) due to deficiency of the common ß-chain (ßc) of the interleukin-3 (IL-3)/IL-5/granulocyte-macrophage colony-stimulating factor (GM-CSF) receptors is a rare monogeneic disease characterized by functional insufficiency of pulmonary macrophages. Hematopoietic stem cell gene therapy for restoring expression of ßc-protein in the hematopoietic system may offer a curative approach. Toward this end, we generated a retroviral construct expressing the murine ßc (mßc) gene and conducted investigations in a murine model of ßc-deficient PAP. Functional correction of mßc activity in mßc-/- bone marrow (BM) cells was demonstrated by restoration of in vitro colony formation in response to GM-CSF. In addition, in a murine in vivo model of mßc-deficient PAP mßc gene transfer to hematopoietic stem cells not only restored the GM-CSF-sensitivity of hematopoietic progenitor cells but also, within a period of 12 weeks, almost completely reversed the morphologic features of surfactant accumulation. These results were obtained despite modest transduction levels (10-20%) and, in comparison to wild-type mice, clearly reduced ßc expression levels were detected in hematopoietic cells. Therefore, our data demonstrating genetic and functional correction of mßc-/- deficiency in vitro as well as in a murine in vivo model of PAP strongly suggest gene therapy as a potential new treatment modality in ßc-deficient PAP.