Subsequent Size Exclusion Chromatography Research Articles

Molar masses and molar mass distributions of commercial regenerated cellulose materials and softwood dissolving pulp determined by SEC/MALLS

The molar masses and molar mass distributions of three commercial regenerated cellulose samples, viscose rayon, Tencel, and Bemliese (or cuprammonium nonwoven), have been determined by dissolution in 8% (w/w) lithium chloride/N,N-dimethylacetamide (LiCl/DMAc) and subsequent size-exclusion chromatography with multi-angle laser-light scattering detection (SEC/MALLS). Before dissolution in LiCl/DMAc, the regenerated cellulose samples were pretreated by the following three methods: (1) soaking in ethylene diamine (EDA) and subsequent solvent exchange to N,N-dimethylacetamide (DMAc) through methanol, (2) soaking in water and subsequent solvent exchange to DMAc through ethanol, and (3) soaking in water and subsequent solvent exchange to tert-butyl alcohol through ethanol and freeze dying. The pretreated samples were dissolved in 8% (w/w) LiCl/DMAc by stirring the cellulose/LiCl/DMAc mixtures for 1–3 weeks followed by dilution to 1% (w/v) LiCl/DMAc for SEC/MALLS analysis. The EDA- and water-pretreated samples gave almost the same SEC-elution pattens and molar mass plots, resulting in similar number- and mass-average molar masses. However, the freeze-dried samples gave 10%‒20% lower mass recovery ratios than those obtained for the EDA- or water-pretreated samples, probably because of incomplete dissolution of the freeze-dried samples in 8% (w/w) LiCl/DMAc. The average mass-average degree of polymerization values of viscose rayon, Tencel, and Bemliese were 340, 530, and 880, respectively. The slopes of the conformation plots were 0.58–0.62, showing that all of the molecules in the three regenerated cellulose samples were dissolved in 1% (w/v) LiCl/DMAc, forming linear random-coil conformations.Graphical abstract

Removal and identification of external protein corona members from RBC‐derived extracellular vesicles by surface manipulating antimicrobial peptides

AbstractIn the last years, extracellular vesicles (EVs), secreted by various cells and body fluids have shown extreme potential in biomedical applications. Increasing number of studies suggest that a protein corona could adhere to the surface of EVs which can have a fundamental effect on their function, targeting and therapeutical efficacy. However, removing and identifying these corona members is currently a challenging task to achieve. In this study we have employed red blood cell‐derived extracellular vesicles (REVs) as a model system and three membrane active antimicrobial peptides (AMPs), LL‐37, FK‐16 and CM15, to test whether they can be used to remove protein corona members from the surface of vesicles. These AMPs were reported to preferentially exert their membrane‐related activity via one of the common helical surface‐covering models and do not significantly affect the interior of lipid bilayer bodies. The interaction between the peptides and the REVs was followed by biophysical techniques, such as flow‐linear dichroism spectroscopy which provided the effective applicable peptide concentration for protein removal. REV samples were then subjected to subsequent size exclusion chromatography and to proteomics analysis. Based on the comparison of control REVs with the peptide treated samples, seventeen proteins were identified as external protein corona members. From the three investigated AMPs, FK‐16 can be considered as the best candidate to further optimize EV‐related applicability of AMPs. Our results on the REV model system envisage that membrane active peptides may become a useful set of tools in engineering and modifying surfaces of EVs and other lipid‐based natural particles.

Cargo-loading of hybrid cowpea chlorotic mottle virus capsids via a co-expression approach

Capsids of the cowpea chlorotic mottle virus (CCMV) are great candidates for the development into in vivo catalytic or therapeutic nanocarriers. However, due to their limited intrinsic stability at physiological pH, thus far no methods exist for incorporating cargo into these nanoparticles in cellulo. Here, we employ a stabilized VW1-VW8 ELP-CCMV variant for the development of a co-expression-based cargo-loading approach. Co-expression of the non-functionalized VW1-VW8 ELP-CCMV coat protein with fusion proteins with enhanced green fluorescent protein (mEGFP) and pyrrolysine synthase D (PylD) in E. coli enabled the purification of cargo-loaded capsids from the bacteria directly either via affinity chromatography or PEG-precipitation and subsequent size exclusion chromatography. Microscopy results indicated that the co-expression does not harm the E. coli cells and that proper folding of the mEGFP domain is not hampered by the co-assembly. Our co-expression strategy is thus a suitable approach to produce cargo-loaded CCMV nanoparticles.

Comparative analysis of tangential flow filtration and ultracentrifugation, both combined with subsequent size exclusion chromatography, for the isolation of small extracellular vesicles.

Small extracellular vesicles (sEVs) provide major promise for advances in cancer diagnostics, prognostics, and therapeutics, ascribed to their distinctive cargo reflective of pathophysiological status, active involvement in intercellular communication, as well as their ubiquity and stability in bodily fluids. As a result, the field of sEV research has expanded exponentially. Nevertheless, there is a lack of standardisation in methods for sEV isolation from cells grown in serum‐containing media. The majority of researchers use serum‐containing media for sEV harvest and employ ultracentrifugation as the primary isolation method. Ultracentrifugation is inefficient as it is devoid of the capacity to isolate high sEV yields without contamination of non‐sEV materials or disruption of sEV integrity. We comprehensively evaluated a protocol using tangential flow filtration and size exclusion chromatography to isolate sEVs from a variety of human and murine cancer cell lines, including HeLa, MDA‐MB‐231, EO771 and B16F10. We directly compared the performance of traditional ultracentrifugation and tangential flow filtration methods, that had undergone further purification by size exclusion chromatography, in their capacity to separate sEVs, and rigorously characterised sEV properties using multiple quantification devices, protein analyses and both image and nano‐flow cytometry. Ultracentrifugation and tangential flow filtration both enrich consistent sEV populations, with similar size distributions of particles ranging up to 200 nm. However, tangential flow filtration exceeds ultracentrifugation in isolating significantly higher yields of sEVs, making it more suitable for large‐scale research applications. Our results demonstrate that tangential flow filtration is a reliable and robust sEV isolation approach that surpasses ultracentrifugation in yield, reproducibility, time, costs and scalability. These advantages allow for implementation in comprehensive research applications and downstream investigations.

Kevlar-like Aramid Polymers from Mixed PET Waste.

This work describes the synthetic approaches, spectroscopic and thermal characterization of aramid polymers prepared from waste polyethylene terephthalate (PET) via sustainable and scalable processes. Direct depolymerization of PET with aliphatic diamines under melt conditions resulted in decomposition without substantial formation of any aramid polymer. The Higashi–Ogata methodology or direct polycondensation of terephthalic acid (TPA) derived from PET waste and p-phenylenediamine, resulted in oligomerization and formation of aramids with a low degree of polymerization. The highest molecular weight polymers were obtained via the acid chloride of TPA, the traditional method. A proprietary solvent enabled the dissolution of most polymers and subsequent size exclusion chromatography analysis in the same solvent. We emphasize that although the soluble polymer compounds are prepared via the traditional route, they are novel. The apparent molecular weights of the soluble polymers ranged between 10–35 kDa (Mn) and 28–81 kDa (Mw). All analogues were prepared with commercially available diamines and diamine combinations. The obtained solid powders were dissolved in D2SO4 and analyzed spectroscopically to qualitatively evaluate the degrees of polymerization, while the solids were characterized via thermogravimetric analysis and differential scanning calorimetry. Many reaction conditions were employed to improve the solution polycondensation reaction, and it was found that addition of pyridine (2 eq) to the NMP reaction medium was crucial in preventing the precipitation of the polymer. Contrary to conventional wisdom, CaCl2 did not play a crucial role in the molecular weight increase of the polymer when oxydianiline was used. Our data indicated that the temperature and absence of CaCl2 provided a boost in molecular weight. Both room temperature and 0 °C reactions generated similar polymers as suggested by nuclear magnetic resonance; however, the cold conditions enhanced gel formation, an important attribute in the future processing of these materials to obtain fibers. All analogues had a high degradation temperature at 5 and 10% weight loss (5% and T10%), above 400 °C, along with high percent char values. A glass transition (Tg) was not detected in any of the analogues prepared.

Recombinant Expression of Alkane-1-Monooxygenase (AlkM) and Catechol 1,2-Dioxygenase (CatA) from Environmental Strains of Acinetobacter baumannii

Genes for the hydrocarbon-degrading enzymes – alkane 1-monooxygenase (AlkM) and catechol 1,2-dioxygenase (CatA) – were amplified from three environmental strains of Acinetobacter baumannii from Pasig River and oil sludge sample. Each gene has been previously cloned into a pBAD/Thio-TOPO plasmid expression vector and the recombinant pBAD/Thio-TOPO plasmids were introduced into E coli TOP10 cells via transformation. This study was conducted to determine if AlkM and CatA proteins will be expressed by the recombinant E coli TOP10 cells, to determine the optimized conditions for protein expression, and to perform purification of expressed AlkM and CatA fusion proteins. The pilot expression resulted in the production of the putative 62 kDa AlkM-thioredoxin and the 50 kDa CatA-thioredoxin fusion proteins, and the identity of the expressed proteins was verified to be AlkM and CatA using western blotting. The study showed that best AlkM and CatA expression was observed when recombinant cells were grown in Super broth using 6-h post-induction time with at least 0.2% arabinose. Purified AlkM and CatA fusion proteins were obtained using affinity chromatography and subsequent size exclusion chromatography with Sephadex G-75 column. The preliminary activity of the purified CatA fusion enzyme observed at 1.128 units/mg was much lower than activities reported for other species, while an AlkM activity assay could not be performed because AlkM fusion proteins from recombinant E coli did not solubilize after purification. These suggest the need to perform an extensive study to address AlkM solubility problem and optimize parameters for enzyme activity of expressed CatA and AlkM. Successful expression of AlkM and Cat A genes from a human bacterial pathogen in non-pathogenic E coli host cells is an initial step to the possible use of recombinant cells in producing AlkM and CatA enzymes from a pathogenic bacterial species in addressing problems of oil contamination

Abstract 2900: PRAMEF2 ubiquitylates and degrades LATS1 leading to enhanced nuclear accumulation of YAP and tumorigenesis

Abstract PRAMEF2 (PRAME Family Member 2) is a Cancer Testis Antigen belonging to the PRAME multi-gene family. All Cancer Testis Antigens are expressed in male testicular germ cells while remaining absent from other adult tissues. Conversely, they show aberrant re-expression in several human malignancies and also elicit immunogenic responses in cancer patients. This makes Cancer Testis Antigens and thereby PRAMEF2, effective and novel targets for cancer vaccine development. However, the physiological function of PRAMEF2 underlying tumorigenesis remains unspecified. Hence, we performed a proteomic screen to explore its unique interactome in the cell. Subsequent size exclusion chromatography and co-immunoprecipitation assays confirmed the role of PRAMEF2 as a BC-box containing substrate recognition subunit of a Cullin 2 based-E3 ubiquitin ligase complex. In the proteomic screen, several proteins were identified, however in-vivo assays revealed LATS1 to be a specific and novel interacting partner of PRAMEF2. LATS1 is a Ser/Thr Kinase of the Hippo Pathway, known to aid tumor suppression. We observed PRAMEF2 mediated ubiquitylation and subsequent proteasomal degradation of LATS1 through ubiquitylation assay. The site for ubiquitylation was mapped to the highly conserved Lysine 860 residue on LATS1. Upon activation of the Hippo Pathway, LATS1 results in phosphorylation of the transcriptional co-activator YAP, leading to its cytosolic translocation. Consequently, PRAMEF2 mediated LATS1 regulation resulted in decreased YAP phosphorylation and its increased accumulation in the nucleus. Furthermore, RTqPCR array and ChIP analysis showed the enhanced expression of YAP target genes implicated in proliferation and metastasis. Moreover, PRAMEF2 depletion also diminished the proliferative capacity, migration potential and invasiveness of tumor cells. Studies in mouse tumor models confirmed that PRAMEF2 abrogates the tumor suppressive function of LATS1 leading to ameliorated tumor growth and metastasis. Taken together, our findings help establish the role of PRAMEF2, an unique Cancer Testis Antigen, as a substrate recognition subunit of a Cullin 2- based E3 ubiquitin ligase complex which ubquitylates and degrades LATS1. Hence, PRAMEF2 realizes its oncogenic potential by increasing YAP nuclear accumulation and promoting tumor cell proliferation and metastasis. Citation Format: Madhurima Ghosh, Sanjeev Das. PRAMEF2 ubiquitylates and degrades LATS1 leading to enhanced nuclear accumulation of YAP and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2900.

Isolation of sesquiterpenoids from Matricaria chamomilla by means of solvent\xa0assisted flavor evaporation and centrifugal partition chromatography

The (semi)volatile fraction of Matricaria chamomilla L., an annual herbal plant from the family of Asteraceae, contains high quantities of sesquiterpenes and sesquiterpenoids. A method was developed to achieve isolation and separation of these compounds, using a combination of solvent assisted flavor evaporation (SAFE) and solid support-free liquid-liquid chromatography. The biphasic liquid solvent system n-heptane/ethyl acetate/methanol/water, 5/2/5/2 v/v/v/v (Arizona S) was elaborated as a suitable solvent system for the simultaneous separation of the target compounds. The lab-scale liquid-liquid chromatography separation performed in a countercurrent chromatography (CCC) column was successfully transferred to a semi-preparative centrifugal partition chromatography (CPC) column, which enabled the isolation of artemisia ketone, artemisia alcohol, α-bisabolone oxide A, and (E)-en-yn-dicycloether. α-Bisabolol oxide A and (Z)-en-yn-dicycloether co-eluted, but were successfully separated by subsequent size-exclusion chromatography (SEC). Similarly, spathulenol and α-bisabolol oxide B were obtained as a mixture, and were separated by means of column chromatography using silica gel as stationary phase. The isolated compounds were characterized by means of nuclear magnetic resonance spectroscopy (NMR) and gas chromatography–mass spectrometry (GC-MS).Graphical abstract

Expanding the Use of Tyrosine Phenol Lyase in the Enzymatic Synthesis of Unnatural Amino Acids

The use of enzymes in the synthesis of pharmaceuticals and biochemical tools offers an attractive approach to generating complex structures, as enzymatic reactions often lead to the formation of specific products in high yields. Tyrosine phenol lyase (TPL), an enzyme biologically involved in the degradation of tyrosine, has been used to synthesize a variety of substituted tyrosines for biochemical studies. Expanding the scope of substrates to naphthol-based compounds or substituted tyrosine-based structures offers an attractive target for enzymatic synthesis of complex molecules which can then be used for in potential pharmaceuticals. Using Citrobacter freundii TPL, we enzymatically synthesized the naphthol-analog of tyrosine, but product was in low yield. The simplest model for the low conversion is that this result suggests the larger substrate is poorly accommodated by the enzyme active site and this poor binding leads to the low yield in the enzymatic reaction. To test this hypothesis, we mutated several bulky residues near the substrate-binding site (M288, M397, F448) to open space for substrate binding and facilitate synthesis of the unnatural amino acid. A series of mutant plasmids were generated using site-directed mutagenesis and sequenced to confirm the mutation. The wild type and mutant enzymes were recombinantly expressed in E. coli and subjected to affinity and ion-exchange chromatography. Although gel electrophoresis of the samples showed a single band and initial enzyme assays with tyrosine showed that the wild type and mutants are active, subsequent size-exclusion chromatography assays suggested the samples contain potential different multimeric forms of the enzyme. As previous studies suggested TPL is tetrameric enzyme, we are currently developing additional purification steps to isolate the tetrameric form of the wild type and mutants. The purified enzyme will then be used to evaluate the effects of the mutations on enzyme activity and substrate specificity. While a goal of this study is aimed at the more efficient production of tyrosine derivatives, a larger goal is to better understand the features important for substrate binding in enzymatic reactions. This understanding may aid in the development of enzymes with a broad range of substrates efficiently converted to products in the biochemical generation of complex molecules.

Structural and binding characterization of the LacdiNAc-specific adhesin (LabA; HopD) exodomain from Helicobacter pylori.

Helicobacter pylori (H. pylori) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA (LacdiNAc-binding adhesin), which is thought to bind specifically to GalNAcβ1-4GlcNAc, occurring in the gastric mucosa. LabA47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 ​Å resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20–49%), LabA shares an L-shaped fold with SabA and BabA. The ‘head’ region contains a 4 ​+ ​3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain (‘crown’ region), the ‘J99-type’ (insertion ~31 ​amino acids), and the H. pylori ‘26695 type’ (insertion ~46 ​amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcβ1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding.

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

A fast and straightforward procedure for vault nanoparticle purification and the characterization of its endocytic uptake

BackgroundVaults are eukaryotic ribonucleoprotein particles composed of up 78 copies of the 97 kDa major vault protein that assembles into a barrel-like, “nanocapsule” enclosing poly(ADP-ribose) polymerase, telomerase-associated protein-1 and small untranslated RNAs. Overall, the molecular mass of vault particles amounts to about 13 MDa. Although it has been implicated in several cellular functions, its physiological roles remain poorly understood. Also, the possibility to exploit it as a nanovector for drug delivery is currently being explored in several laboratories. MethodsUsing the baculovirus expression system, vaults were expressed and purified by a dialysis step using a 1 MDa molecular weight cutoff membrane and a subsequent size exclusion chromatography. Purity was assessed by SDS-PAGE, transmission electron microscopy and dynamic light scattering. Particle's endocytic uptake was monitored by flow cytometry and confocal microscopy. ResultsThe purification protocol here reported is far simpler and faster than those currently available and lead to the production of authentic vault. We then demonstrated its clathrin-mediated endocytic uptake by normal fibroblast and glioblastoma, but not carcinoma cell lines. In contrast, no significant caveolin-mediated endocytosis was detected. ConclusionsThese results provide the first evidence for an intrinsic propensity of the vault complex to undergo endocytic uptake cultured eukaryotic cells. General significanceThe newly developed purification procedure will greatly facilitate any investigation based on the use of the vault particle as a natural nanocarrier. Its clathrin-mediated endocytic uptake observed in normal and in some tumor cell lines sheds light on its physiological role.

Comparison of different methods for MP detection: What can we learn from them, and why asking the right question before measurements matters?

In recent years, an increasing trend towards investigating and monitoring the contamination of the environment by microplastics (MP) (plastic pieces < 5 mm) has been observed worldwide. Nonetheless, a reliable methodology that would facilitate and automate the monitoring of MP is still lacking. With the goal of selecting practical and standardized methods, and considering the challenges in microplastics detection, we present here a critical evaluation of two vibrational spectroscopies, Raman and Fourier transform infrared (FTIR) spectroscopy, and two extraction methods: thermal extraction desorption gas chromatography mass spectrometry (TED-GC-MS) and liquid extraction with subsequent size exclusion chromatography (SEC) using a soil with known contents of PE, PP, PS and PET as reference material. The obtained results were compared in terms of measurement time, technique handling, detection limits and requirements for sample preparation. The results showed that in designing and selecting the right methodology, the scientific question that determines what needs to be understood is significant, and should be considered carefully prior to analysis. Depending on whether the object of interest is quantification of the MP particles in the sample, or merely a quick estimate of sample contamination with plastics, the appropriate method must be selected. To obtain overall information about MP in environmental samples, the combination of several parallel approaches should be considered.

Wavelength-Dependent Photochemistry of Oxime Ester Photoinitiators

The design of efficient radical photoinitiating systems requires a systematic and detailed evaluation of their photochemical characteristics. Correlating absorbance and the corresponding electronic transitions of a photoinitiator is critical for understanding its photoinduced reaction pathways. In the current contribution, we provide an in-depth investigation into the photochemistry and photophysics of two oxime ester derivatives (O-benzoyl-α-oxooxime, OXE01, and O-acetyloxime, OXE02), known for their excellent performance in pigmented formulations. In particular, we shed light on their wavelength-dependent photopolymerization properties. We utilized a combination of UV–vis spectroscopy, density functional theory (DFT) calculations, photochemically induced dynamic nuclear polarization spectroscopy (photo-CIDNP), and pulsed-laser polymerization with a wavelength-tunable laser with subsequent size exclusion chromatography coupled to high-resolution electrospray ionization mass spectrometry (PLP-SEC-ESI-MS) ...

Systematic Assessment of the Photochemical Stability of Photoinitiator-Derived Macromolecular Chain Termini

The photostability of polymeric materials is crucial for their applicability, especially under potentially harsh environmental conditions. In the current study, the influence of methyl-substitution on the photochemical stability of photoinitiator-derived benzoyl end groups is systematically investigated by a combination of pulsed-laser polymerization and subsequent size exclusion chromatography coupled with electrospray ionization mass spectrometry (PLP–SEC–ESI–MS), chemically induced dynamic nuclear polarization–nuclear magnetic resonance spectroscopy (CIDNP–NMR), and density functional theory (DFT) calculations. Poly(methyl methacrylate)s (pMMA) were synthesized employing benzoin-type photoinitiators with systematically substituted benzoyl moieties (i.e., 2-methylbenzoin, 3-methylbenzoin, 4-methylbenzoin, 2,4-dimethylbenzoin, 2,6-dimethylbenzoin, 2,4,6-trimethylbenzoin, 2,3,5,6-tetramethylbenzoin, and 2,3,4,5,6-pentamethylbenzoin). Photoinduced cleavage of the photoinitiator-based end group (irradiation...

Expression and purification of functional human glycogen synthase-1:glycogenin-1 complex in insect cells

We report the successful expression and purification of functional human muscle glycogen synthase (GYS1) in complex with human glycogenin-1 (GN1). Stoichiometric GYS1:GN1 complex was produced by co-expression of GYS1 and GN1 using a bicistronic pFastBac™-Dual expression vector, followed by affinity purification and subsequent size-exclusion chromatography. Mass spectrometry analysis identified that GYS1 is phosphorylated at several well-characterised and uncharacterised Ser/Thr residues. Biochemical analysis, including activity ratio (in the absence relative to that in the presence of glucose-6-phosphate) measurement, covalently attached phosphate estimation as well as phosphatase treatment, revealed that recombinant GYS1 is substantially more heavily phosphorylated than would be observed in intact human or rodent muscle tissues. A large quantity of highly-pure stoichiometric GYS1:GN1 complex will be useful to study its structural and biochemical properties in the future, which would reveal mechanistic insights into its functional role in glycogen biosynthesis.

Novel Ubiquitin-derived High Affinity Binding Proteins with Tumor Targeting Properties

Targeting effector molecules to tumor cells is a promising mode of action for cancer therapy and diagnostics. Binding proteins with high affinity and specificity for a tumor target that carry effector molecules such as toxins, cytokines, or radiolabels to their intended site of action are required for these applications. In order to yield high tumor accumulation while maintaining low levels in healthy tissues and blood, the half-life of such conjugates needs to be in an optimal range. Scaffold-based binding molecules are small proteins with high affinity and short systemic circulation. Due to their low molecular complexity, they are well suited for combination with effector molecules as well as half-life extension technologies yielding therapeutics with half-lives adapted to the specific therapy. We have identified ubiquitin as an ideal scaffold protein due to its outstanding biophysical and biochemical properties. Based on a dimeric ubiquitin library, high affinity and specific binding molecules, so-called Affilin® molecules, have been selected against the extradomain B of fibronectin, a target almost exclusively expressed in tumor tissues. Extradomain B-binding molecules feature high thermal and serum stability as well as strong in vitro target binding and in vivo tumor accumulation. Application of several half-life extension technologies results in molecules of largely unaffected affinity but significantly prolonged in vivo half-life and tumor retention. Our results demonstrate the utility of ubiquitin as a scaffold for the generation of high affinity binders in a modular fashion, which can be combined with effector molecules and half-life extension technologies.

Nuclear RNase P of Trypanosoma brucei: A Single Protein in Place of the Multicomponent RNA-Protein Complex

SummaryRNase P is the endonuclease that removes 5′ extensions from tRNA precursors. In its best-known form, the enzyme is composed of a catalytic RNA and a protein moiety variable in number and mass. This ribonucleoprotein enzyme is widely considered ubiquitous and apparently reached its highest complexity in the eukaryal nucleus, where it is typically composed of at least ten subunits. Here, we show that in the protist Trypanosoma brucei, two proteins are the sole forms of RNase P. They localize to the nucleus and the mitochondrion, respectively, and have RNase P activity each on their own. The protein-RNase P is, moreover, capable of replacing nuclear RNase P in yeast cells. This shows that complex ribonucleoprotein structures and RNA catalysis are not necessarily required to support tRNA 5′ end formation in eukaryal cells.

Dual Role of the Molybdenum Cofactor Biosynthesis Protein MOCS3 in tRNA Thiolation and Molybdenum Cofactor Biosynthesis in Humans

We studied two pathways that involve the transfer of persulfide sulfur in humans, molybdenum cofactor biosynthesis and tRNA thiolation. Investigations using human cells showed that the two-domain protein MOCS3 is shared between both pathways. MOCS3 has an N-terminal adenylation domain and a C-terminal rhodanese-like domain. We showed that MOCS3 activates both MOCS2A and URM1 by adenylation and a subsequent sulfur transfer step for the formation of the thiocarboxylate group at the C terminus of each protein. MOCS2A and URM1 are β-grasp fold proteins that contain a highly conserved C-terminal double glycine motif. The role of the terminal glycine of MOCS2A and URM1 was examined for the interaction and the cellular localization with MOCS3. Deletion of the C-terminal glycine of either MOCS2A or URM1 resulted in a loss of interaction with MOCS3. Enhanced cyan fluorescent protein and enhanced yellow fluorescent protein fusions of the proteins were constructed, and the fluorescence resonance energy transfer efficiency was determined by the decrease in the donor lifetime. The cellular localization results showed that extension of the C terminus with an additional glycine of MOCS2A and URM1 altered the localization of MOCS3 from the cytosol to the nucleus.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in &lt;em&gt;Saccharomyces cerevisiae&lt;/em&gt;

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be >90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.