Cloning, expression and purification of the cytokine FLT3-ligand for protein interaction studies

Soluble Form ◽

Spectrometry Analysis ◽

Interaction Studies ◽

Proliferation And Differentiation ◽

Culture Models ◽

Full Length Sequence ◽

The FLT3-FLT3L receptor complex is responsible for the proliferation and differentiation of hematopoietic progenitor and stem cells, and is involved in several hematological malignancies. A truncated, soluble form of FLT3L was amplified from the full-length sequence by PCR and cloned into a genomic integration and expression vector for Kluyveromyces lactis. Expression and secretion of the 161 amino acid FLT3L was analyzed by SDS-PAGE and Western blot and showed smeared bands around 50kDa consistent with a high level of varying glycosylation as expected. A yield of 100ug/mL of culture was obtained. Purification was performed by Ni-affinity chromatography and the protein was confirmed to be in the elution fraction via mass spectrometry analysis. Purified FLT3L was able to activate RAW264.7 cells after 24hr incubation period causing large vacuoles and a dendritic appearance. FLT3L will be utilized for LARC protein interaction studies to search for interactors that might serve as therapeutic agents in human body fluids and cell culture models of myeloid leukemia.

Cloning, expression and purification of the cytokine FLT3-ligand for protein interaction studies

10.32920/ryerson.14656716.v1 ◽

2021 ◽

Author(s):

Lauren Alicia Agro

Keyword(s):

Protein Interaction ◽

Kluyveromyces Lactis ◽

Soluble Form ◽

Spectrometry Analysis ◽

Interaction Studies ◽

Proliferation And Differentiation ◽

Culture Models ◽

Full Length Sequence ◽

Neuronal Proteomic Analysis of the Ubiquitinated Substrates of the Disease-Linked E3 Ligases Parkin and Ube3a

BioMed Research International ◽

10.1155/2018/3180413 ◽

2018 ◽

Vol 2018 ◽

pp. 1-14 ◽

Cited By ~ 6

Author(s):

Aitor Martinez ◽

Juanma Ramirez ◽

Nerea Osinalde ◽

Jesus M. Arizmendi ◽

Ugo Mayor

Keyword(s):

Proteasome Inhibitors ◽

Brain Dysfunction ◽

E3 Ligases ◽

Spectrometry Analysis ◽

Ubiquitinated Proteins ◽

Neuronal Proteins ◽

Culture Models ◽

First Time ◽

Cell Culture Models

Both Parkin and UBE3A are E3 ubiquitin ligases whose mutations result in severe brain dysfunction. Several of their substrates have been identified using cell culture models in combination with proteasome inhibitors, but not in more physiological settings. We recently developed theUbbiostrategy to isolate ubiquitinated proteins in flies and have now identified by mass spectrometry analysis the neuronal proteins differentially ubiquitinated by those ligases. This is an example of how flies can be used to provide biological material in order to reveal steady state substrates of disease causing genes. Collectively our results provide new leads to the possible physiological functions of the activity of those two disease causing E3 ligases. Particularly, in the case of Parkin the novelty of our data originates from the experimental setup, which is not overtly biased by acute mitochondrial depolarisation. In the case of UBE3A, it is the first time that a nonbiased screen for its neuronal substrates has been reported.

The Variability of Thymol and Carvacrol Contents Reveals the Level of Antibacterial Activity of the Essential Oils from Different Accessions of Oliveria decumbens

Antibiotics ◽

10.3390/antibiotics9070409 ◽

2020 ◽

Vol 9 (7) ◽

pp. 409

Author(s):

Tahereh Khoshbakht ◽

Akbar Karami ◽

Aminallah Tahmasebi ◽

Filippo Maggi

Keyword(s):

Antibacterial Activity ◽

Essential Oils ◽

Positive Control ◽

Human Pathogens ◽

Clavibacter Michiganensis ◽

Spectrometry Analysis ◽

High Level ◽

Shed Light ◽

Chromatographic Mass

Oliveria decumbens (Apiaceae) is an aromatic herb traditionally employed in the Persian medicine for the treatment of infectious and gastrointestinal disorders. In the present study, we analyzed the chemical composition of essential oils obtained from different Iranian populations and evaluated their efficacy on a panel of human pathogens (Staphylococcus aureus and Escherichia coli), probiotic (Bacillus subtilis), and phytopathogens (Clavibacter michiganensis, Curtobacterium flaccumfaciens, Xanthomonas citri, and Agrobacterium tumefaciens). The gas chromatographic-mass spectrometry analysis put in evidence four main volatile constituents such as thymol (20.3–36.4%), carvacrol (18.8–33.1%), γ-terpinene (10.6–25.9%), and p-cymene (9.5–17.3%), though with significant variability from an essential oil to another. Notably, the oils from the populations sited in Nourabad Mamasani and Dehdasht showed the highest amount of the phenolic monoterpenes thymol (36.4 and 35.2%, respectively) and carvacrol (33.1 and 30.6%, respectively). The antibacterial activity of O. decumbens essential oils was assessed by the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods, showing high activity for the samples from Nourabad Mamasani and Dehdasht populations exhibiting high level of the above phenolics. The obtained MIC and MBC values (mg/ml) were in the ranges 0.0625–2 mg/ml and 1–16 mg/ml, respectively. Noteworthy, in some cases, the antibacterial activity of O. decumbens essential oils was higher than that of chloramphenicol used as positive control. The average MBCs displayed by the O. decumbens samples showed that C. flaccumfaciens had the highest sensitivity to the essential oils. Based on these results, our work shed light on selected O. decumbens populations deserving proper breeding and cultivation strategies in order to warrantee production of bioactive essential oils to be used at pharmaceutical and agricultural level to combat several pathogens.

Role of lgtC in Resistance of Nontypeable Haemophilus influenzae Strain R2866 to Human Serum

Infection and Immunity ◽

10.1128/iai.00722-06 ◽

2006 ◽

Vol 74 (11) ◽

pp. 6226-6235 ◽

Cited By ~ 35

Author(s):

Alice L. Erwin ◽

Simon Allen ◽

Derek K. Ho ◽

Paul J. Bonthius ◽

Justin Jarisch ◽

...

Keyword(s):

Haemophilus Influenzae ◽

Human Serum ◽

Start Codon ◽

Enzyme Linked Immunosorbent Assay ◽

Serum Resistance ◽

Nontypeable Haemophilus Influenzae ◽

Spectrometry Analysis ◽

Nthi Strain ◽

ABSTRACT We are investigating a nontypeable Haemophilus influenzae (NTHI) strain, R2866, isolated from a child with meningitis. R2866 is unusually resistant to killing by normal human serum. The serum 50% inhibitory concentration (IC50) for this strain is 18%, approaching that of encapsulated H. influenzae. R3392 is a derivative of R2866 that was found to have increased sensitivity to human serum (IC50, 1.5%). Analysis of tetrameric repeat regions within lipooligosaccharide (LOS) biosynthetic genes in both strains indicated that the glycosyltransferase gene lgtC was out of frame (“off”) in most colonies of R3392 but in frame with its start codon (“on”) in most colonies of the parent. We sought antigenic and biochemical evidence for modification of the LOS structure. In a whole-cell enzyme-linked immunosorbent assay, strain R3392 displayed reduced binding of the Galα1,4Gal-specific monoclonal antibody 4C4. Mass spectrometry analysis of LOS from strain R2866 indicated that the primary oligosaccharide glycoform contained four heptose and four hexose residues, while that of R3392 contained four heptose and three hexose residues. We conclude that the R2866 lgtC gene encodes a galactosyltransferase involved in synthesis of the 4C4 epitope, as in other strains, and that expression of lgtC is associated with the high-level serum resistance that has been observed for this strain. This is the first description of the genetic basis of high-level serum resistance in NTHI, as well as the first description of LOS composition in an NTHI strain for which the complete genome sequence has been determined.

Nonspecific Cleavages Arising from Reconstitution of Trypsin under Mildly Acidic Conditions

10.1101/2020.05.26.116509 ◽

2020 ◽

Author(s):

Ben Niu ◽

Michael Martinelli ◽

Yang Jiao ◽

Eric Meinke ◽

Mingyan Cao ◽

...

Keyword(s):

Storage Conditions ◽

Tryptic Digestion ◽

Spectrometry Analysis ◽

Product Characterization ◽

Proteomics Research ◽

Acidic Conditions ◽

High Level ◽

The Impact ◽

And Storage

AbstractTryptic digestion of proteins followed by liquid chromatography with tandem mass spectrometry analysis is an extensively used approach in proteomics research and biopharmaceutical product characterization, owing to the high level of cleavage fidelity produced with this technique. However, nonspecific trypsin cleavages have been frequently reported and shown to be related to a number of digestion conditions and predigestion sample treatments. In this work, we reveal that, for a number of commercial trypsins, reconstitution and storage conditions can have a significant impact on the occurrence of trypsin nonspecific cleavages. We analyzed the tryptic digestion of a variety of biotherapeutics, using trypsins reconstituted under different conditions. The results indicate that, for many commercial trypsins, commonly recommended reconstitution/storage conditions (mildly acidic, e.g., 50 mM acetic acid, 1 mM HCl) can actually promote nonspecific trypsin activities, which are time dependent and can be as high as 20% in total relative abundance. In contrast, using water for reconstitution and storage can effectively limit nonspecific cleavages to 1%. Interestingly, the performances of different commercial trypsins were found to be quite distinct in their levels of nonspecific cleavages and responses to the two reconstitution conditions. Our findings demonstrate the importance of choosing the appropriate trypsin for tryptic digestion and the necessity of assessing the impact of trypsin reconstitution and storage on nonspecific cleavages. We advocate for manufacturers of commercial trypsins to reevaluate manufacturing processes and reconstitution/storage conditions to provide good cleavage specificity.

Unique Protein Interaction Networks Define The Chromatin Remodeling Module of The NuRD Complex

10.1101/2021.01.27.428018 ◽

2021 ◽

Author(s):

Mehdi Sharifi Tabar ◽

Caroline Giardina ◽

Yue Julie Feng ◽

Habib Francis ◽

Hakimeh Moghaddas Sani ◽

...

Keyword(s):

Chromatin Remodeling ◽

Protein Interaction ◽

Treatment Strategies ◽

Interaction Network ◽

Terminal Segment ◽

Nurd Complex ◽

Nucleosome Remodeling ◽

Spectrometry Analysis ◽

Underlying Mechanisms

AbstractThe combination of four proteins and their paralogues including MBD2/3, GATAD2A/B, CDK2AP1, and CHD3/4/5, which we refer to as the MGCC module, form the chromatin remodeling module of the Nucleosome Remodeling and Deacetylase (NuRD) complex, a gene repressor complex. Specific paralogues of the MGCC subunits such as MBD2 and CHD4 are amongst the key repressors of adult-stage fetal globin and provide important targets for molecular therapies in beta (β)-thalassemia. However, mechanisms by which the MGCC module acquires paralogue-specific function and specificity have not been addressed to date. Understanding the protein-protein interaction (PPI) network of the MGCC subunits is essential in defining underlying mechanisms and developing treatment strategies. Therefore, using pulldown followed by mass spectrometry analysis (PD-MS) we report a proteome-wide interaction network of the MGCC module in a paralogue-specific manner. Our data also demonstrate that the disordered C-terminal region of CHD3/4/5 is a gateway to incorporate remodeling activity into both the ChAHP (CHD4, ADNP, HP1γ) and NuRD complexes in a mutually exclusive manner. We define a short aggregation prone region (APR) within the C-terminal segment of GATAD2B that is essential for the interaction of CHD4 and CDK2AP1 with the NuRD complex. Finally, we also report an association of CDK2AP1 with the Nuclear Receptor Co-Repressor (NCOR) complex. Overall, this study provides insight into the possible mechanisms through which the MGCC module can achieve specificity and diverse biological functions.

Expression and Purification of Soluble Recombinant Gsttagged Bpsl2774 Protein from Burkholderia Pseudomallei K96243

IIUM Medical Journal Malaysia ◽

10.31436/imjm.v16i1.1146 ◽

2017 ◽

Vol 16 (1) ◽

Author(s):

Aisyah Mohamed Rehan ◽

Hanisah Ujang ◽

Siti Marhamah Drahaman ◽

Nor Azurah Mat Akhir ◽

Noraslinda Muhamad Bunnori ◽

...

Keyword(s):

Affinity Chromatography ◽

Burkholderia Pseudomallei ◽

Insertion Site ◽

Plasmid Vector ◽

Target Protein ◽

Soluble Form ◽

Spectrometry Analysis ◽

E Coli ◽

Sufficient Concentration

Introduction: Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease endemic in Southeast Asia and northern Australia. Cases have been reported in Pahang, Johor Bahru and Kedah. The disease is difficult to combat as B. pseudomallei has shown resistance to various antibiotics and much is still not understood about its pathogenicity. It is suggested that investigating the bacterium hypothetical proteins may provide potential new targets for the development of antimicrobials. The gene of interest in this study, BPSL2774, encoding BPSL2774 hypothetical protein, is a target gene that was predicted as essential using transposon-directed insertion site sequencing technique (TraDIS). We aimed to express and purify soluble GST-tagged BPSL2774 protein at sufficient concentration for future functional assays. Materials and method: The BPSL2774 gene has previously been amplified from genomic DNA of B. pseudomallei K96243 and cloned into pDEST15 (GST-tag) plasmid vector. In this work, the clone was transformed into E. coli BL21(DE3) expression strain cells for up-scaled protein preparations in 0.5 L and 1 L cultures. The auto-induction method was adopted for protein expression. GST-tag affinity chromatography was performed for protein purification and the fractions obtained were analyzed using SDS-PAGE. Results: The target protein was successfully expressed in soluble form and its highest concentration from a 0.8 mL elution fraction was at 1.38 mg/mL. Mass spectrometry analysis of 60 kDa coomassie-stained gel band cut confirmed the presence of the soluble expressed target protein, co-purified with E. coli chaperonin proteins, possibly due to their interaction with the target protein. Higher purity can be achieved through further purification steps following initial GST-tag affinity chromatography. Conclusion: The purified protein was at an acceptable purity and at sufficient concentration for use as samples in a glycosyltransferase bioluminescence assay in the near future.

Mass spectrometry analysis of the photosystem II assembly factor Psb27 revealed variations in its lipid modification

Photosynthesis Research ◽

10.1007/s11120-021-00891-7 ◽

2021 ◽

Author(s):

Jan Lambertz ◽

Pasqual Liauw ◽

Julian P. Whitelegge ◽

Marc M. Nowaczyk

Keyword(s):

Mass Spectrometry ◽

Photosystem Ii ◽

Protein Complexes ◽

Exchange Chromatography ◽

Large Network ◽

Spectrometry Analysis ◽

Assembly Factor ◽

Membrane Protein Complexes ◽

AbstractThe assembly of large, multi-cofactor membrane protein complexes like photosystem II (PSII) requires a high level of coordination. The process is facilitated by a large network of auxiliary proteins that bind transiently to unassembled subunits, preassembled modules or intermediate states of PSII, which are comprised of a subset of subunits. However, analysis of these immature, partially assembled PSII complexes is hampered by their low abundance and intrinsic instability. In this study, PSII was purified from the thermophilic cyanobacterium Thermosynechococcus elongatus via Twin-Strep-tagged CP43 and further separated by ion exchange chromatography into mature and immature complexes. Mass spectrometry analysis of the immature Psb27-PSII intermediate revealed six different Psb27 proteoforms with distinct lipid modifications. The maturation and functional role of thylakoid localized lipoproteins are discussed.

Coidentification of mcr-4.3 and blaNDM-1 in a Clinical Enterobacter cloacae Isolate from China

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00649-18 ◽

2018 ◽

Vol 62 (10) ◽

Cited By ~ 17

Author(s):

Bhakti Chavda ◽

Jingnan Lv ◽

Mengyun Hou ◽

Kalyan D. Chavda ◽

Barry N. Kreiswirth ◽

...

Keyword(s):

Lipid A ◽

Susceptibility Testing ◽

Enterobacter Cloacae ◽

Ionization Mass ◽

The Novel ◽

Laser Desorption Ionization ◽

Content Type ◽

Spectrometry Analysis ◽

ABSTRACT We describe the first report of a clinical colistin-resistant ST84 Enterobacter cloacae isolate coharboring mcr-4.3 (previously named mcr-4.2) and blaNDM-1 from a patient in China. The blaNDM-1-harboring IncX3 plasmid and the novel mcr-4.3-harboring ColE plasmid were completely sequenced. Although this isolate showed a high level of resistance to colistin, mcr-4.3 plasmid transformation, gene subcloning, susceptibility testing, and lipid A matrix-assisted laser desorption ionization mass spectrometry analysis indicated that mcr-4.3 itself does not confer resistance to colistin.