scholarly journals Engineering and characterising a novel, highly potent bispecific antibody iMab-CAP256 that targets HIV-1

Retrovirology ◽  
2019 ◽  
Vol 16 (1) ◽  
Author(s):  
Tumelo Moshoette ◽  
Stuart Alvaro Ali ◽  
Maria Antonia Papathanasopoulos ◽  
Mark Andrew Killick
Keyword(s):  
Clinical Performance ◽  
Recombinant Expression ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
T Antigen ◽  
Size Exclusion ◽  
Comparable Efficacy ◽  
Antibody Molecule ◽  
Parental Antibody ◽  
Hiv 1

Abstract The existing repertoire of HIV-1 patient derived broadly neutralising antibodies (bNAbs) that target the HIV-1 envelope glycoprotein (Env) present numerous and exciting opportunities for immune-based therapeutic and preventative strategies against HIV-1. Combination antibody therapy is required to ensure greater neutralization coverage and limit Env mediated escape mutations following treatment pressure. Engineered bispecific bNAbs (bibNAbs) assimilate the advantages of combination therapy into a single antibody molecule with several configurations reporting potency enhancement as a result of the increased avidity and simultaneous engagement of targeted epitopes. We report the engineering of a novel bibNAb (iMab-CAP256) comprising the highly potent, CAP256.VRC26.25 bNAb with anticipated extension in neutralization coverage through pairing with the host directed, anti-CD4 antibody, ibalizumab (iMab). Recombinant expression of parental monoclonal antibodies and the iMab-CAP256 bibNAb was performed in HEK293T (Human embryonic kidney 293 T antigen) cells, purified to homogeneity by Protein-A affinity chromatography followed by size exclusion chromatography. Antibody assembly and binding functionality of Fab moieties was confirmed by SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) and ELISA, respectively. Breadth and potency were evaluated against a geographical diverse HIV-1 pseudovirus panel (n = 20). Overall, iMab-CAP256 demonstrated an expanded neutralizing coverage, neutralizing single, parental antibody resistant pseudovirus strains and an enhanced neutralization potency against all dual sensitive strains (average fold increase over the more potent parental antibody of 11.4 (range 2 to 31.8). Potency enhancement was not observed for the parental antibody combination treatment (iMab + CAP256) suggesting the presence of a synergistic relationship between the CAP256 and iMab paratope combination in this bibNAb configuration. In addition, iMab-CAP256 bibNAbs exhibited comparable efficacy to other bibNAbs PG9-iMab and 10E08-iMab previously reported in the literature. The enhanced neutralization coverage and potency of iMAb-CAP256 over the parental bNAbs should facilitate superior clinical performance as a therapeutic or preventative strategy against HIV-1.

Phospholipase C (heat-labile hemolysin) of Pseudomonas aeruginosa: purification and preliminary characterization

1982 ◽  
Vol 152 (1) ◽  
pp. 239-245
Author(s):  
R M Berka ◽  
M L Vasil
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Phospholipase C ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Tetradecyltrimethylammonium Bromide ◽  
Heat Labile ◽  
Hydrophobic Moiety ◽  
Culture Supernatants

Phospholipase C (heat-labile hemolysin) was purified from Pseudomonas aeruginosa culture supernatants to near homogeneity by ammonium sulfate precipitation followed by a novel application of DEAE-Sephacel chromatography. Enzymatic activity remained associated with DEAE-Sephacel even in the presence of 1 M NaCl, but was eluted with a linear gradient of 0 to 5% tetradecyltrimethylammonium bromide. Elution from DEAE-Sephacel was also obtained with 2% lysophosphatidylcholine, and to a lesser extent with 2% phosphorylcholine, but not at all with choline. The enzyme was highly active toward phospholipids possessing substituted ammonium groups (e.g., phosphatidycholine, lysophosphatidylcholine, and sphingomyelin); however, it had little if any activity toward phospholipids lacking substituted ammonium groups (e.g., phosphatidylethanolamine, phosphatidylserine, and phosphaditylglycerol). Collectively, these data suggest that phospholipase C from P. aeruginosa exhibits high affinity for substituted ammonium groups, but requires an additional hydrophobic moiety for optimum binding. The specific activity of the purified enzyme preparation increased 1,900-fold compared with that of culture supernatants. The molecular weight of the phospholipase C was estimated to be 78,000 by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephacryl S-200 column chromatography and was 76,000 by high-performance size exclusion chromatography. The isoelectric point was 5.5. Amino acid analysis showed that phospholipase C was rich in glycine, serine, threonine, aspartyl, glutamyl, and aromatic amino acids, but was cystine free.

scholarly journals Comparison of the Proteosomes and Antigenicities of Secreted and Cellular Proteins Produced by Mycobacterium paratuberculosis

2006 ◽  
Vol 13 (10) ◽  
pp. 1155-1161 ◽  
Author(s):  
Donghee Cho ◽  
Michael T. Collins
Keyword(s):  
Solid Phase ◽  
Expression Profiles ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Size Exclusion ◽  
Enzyme Linked Immunosorbent Assay ◽  
Infected Cattle ◽  
Mycobacterium Paratuberculosis ◽  
Serologic Tests ◽  
Sds Page

ABSTRACT The protein expression profiles and antigenicities of both culture filtrates (CF) and cellular extracts (CE) of Mycobacterium paratuberculosis were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), one-dimensional electrophoresis (1-DE) and 2-DE immunoblotting, and enzyme-linked immunosorbent assay (ELISA). The CF proteins were harvested from supernatants of stationary-phase liquid cultures and concentrated by size exclusion filtration. The CE proteins were extracted by mechanical disruption of cells using glass beads and a high-speed agitator. Analysis of SDS-PAGE gels showed that the majority of CF proteins had low molecular masses (<50 kDa), whereas CE protein mass ranged more evenly over a broader range up to 100 kDa. By 2-DE, CF proteins had a narrow array of pI values, with most being between pH 4.0 and 5.5; CE proteins spanned pI values from pH 4.0 to 7.0. The antigenicities of CF and CE proteins were first determined by 1-DE and 2-DE immunoblotting with serum from a cow naturally infected with M. paratuberculosis. The serum reacted strongly to more proteins in the CF than the CE. Sera from 444 infected and 412 uninfected cattle were tested by ELISA with CF and CE as solid-phase antigens. Receiver-operator characteristic curve analysis of the ELISA results showed a significantly greater area under the curve for CF compared to CE (P < 0.05). A high degree of variability in protein binding patterns was shown with 1-DE immunoblot analysis with 31 sera from M. paratuberculosis-infected cattle. Collectively, these results indicate that serologic tests for bovine paratuberculosis may be improved by using proteins derived from CF instead of CE. To maximize the diagnostic sensitivity of serologic tests, multiple proteins will be required. Even so, a CF ELISA may not be able to detect all M. paratuberculosis-infected cattle, in particular those in the early stages of infection that have yet to mount an antibody response.

scholarly journals Impact of Botrytis cinerea Contamination on the Characteristics and Foamability of Yeast Macromolecules Released during the Alcoholic Fermentation of a Model Grape Juice

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 472
Author(s):  
Richard Marchal ◽  
Thomas Salmon ◽  
Ramon Gonzalez ◽  
Belinda Kemp ◽  
Céline Vrigneau ◽  
...  
Keyword(s):  
Botrytis Cinerea ◽  
Alcoholic Fermentation ◽  
Periodic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Grape Juice ◽  
Size Exclusion ◽  
Periodic Acid Schiff ◽  
Exclusion Chromatography ◽  
The Impact

Botrytis cinerea is a fungal pathogen responsible for the decrease in foamability of sparkling wines. The proteolysis of must proteins originating from botrytized grapes is well known, but far less information is available concerning the effect of grape juice contamination by Botrytis. The impact from Botrytis on the biochemical and physico-chemical characteristics of proteins released from Saccharomyces during alcoholic fermentation remains elusive. To address this lack of knowledge, a model grape juice was inoculated with three enological yeasts with or without the Botrytis culture supernatant. Size exclusion chromatography coupled to multi-angle light scattering (SEC-MALLS) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) techniques (AgNO3 and periodic acid Schiff staining) was used in the study. When Botrytis enzymes were present, a significant degradation of the higher and medium MW molecules released by Saccharomyces was observed during alcoholic fermentation whilst the lower MW fraction increased. For the three yeast strains studied, the results clearly showed a strong decrease in the wine foamability when synthetic musts were inoculated with 5% (v/v) of Botrytis culture due to fungus proteases.

scholarly journals Purification and Characterization of a Novel Mannitol Dehydrogenase from a Newly Isolated Strain of Candida magnoliae

2003 ◽  
Vol 69 (8) ◽  
pp. 4438-4447 ◽  
Author(s):  
Jung-Kul Lee ◽  
Bong-Seong Koo ◽  
Sang-Yong Kim ◽  
Hyung-Hwan Hyun
Keyword(s):  
Substrate Specificity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Catalytic Efficiency ◽  
Industrial Applications ◽  
Amino Acid Sequences ◽  
Size Exclusion ◽  
Mannitol Dehydrogenase ◽  
High Substrate ◽  
Candida Magnoliae

ABSTRACT Mannitol biosynthesis in Candida magnoliae HH-01 (KCCM-10252), a yeast strain that is currently used for the industrial production of mannitol, is catalyzed by mannitol dehydrogenase (MDH) (EC 1.1.1.138). In this study, NAD(P)H-dependent MDH was purified to homogeneity from C. magnoliae HH-01 by ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The relative molecular masses of C. magnoliae MDH, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion chromatography, were 35 and 142 kDa, respectively, indicating that the enzyme is a tetramer. This enzyme catalyzed both fructose reduction and mannitol oxidation. The pH and temperature optima for fructose reduction and mannitol oxidation were 7.5 and 37°C and 10.0 and 40°C, respectively. C. magnoliae MDH showed high substrate specificity and high catalytic efficiency (k cat = 823 s−1, K m = 28.0 mM, and k cat /K m = 29.4 mM−1 s−1) for fructose, which may explain the high mannitol production observed in this strain. Initial velocity and product inhibition studies suggest that the reaction proceeds via a sequential ordered Bi Bi mechanism, and C. magnoliae MDH is specific for transferring the 4-pro-S hydrogen of NADPH, which is typical of a short-chain dehydrogenase reductase (SDR). The internal amino acid sequences of C. magnoliae MDH showed a significant homology with SDRs from various sources, indicating that the C. magnoliae MDH is an NAD(P)H-dependent tetrameric SDR. Although MDHs have been purified and characterized from several other sources, C. magnoliae MDH is distinguished from other MDHs by its high substrate specificity and catalytic efficiency for fructose only, which makes C. magnoliae MDH the ideal choice for industrial applications, including enzymatic synthesis of mannitol and salt-tolerant plants.

scholarly journals A T cell receptor V alpha domain expressed in bacteria: does it dimerize in solution?

1996 ◽  
Vol 184 (4) ◽  
pp. 1251-1258 ◽  
Author(s):  
D Plaksin ◽  
S Chacko ◽  
P McPhie ◽  
A Bax ◽  
E A Padlan ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
T Cell Activation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Receptor ◽  
Size Exclusion ◽  
Resonance Spectroscopy ◽  
Gel Chromatography ◽  
Mhc Class I Molecule

To evaluate the potential for dimerization through a particular T cell receptor (TCR) domain, we have cloned the cDNA encoding a TCR V alpha from a hybridoma with specificity for the human immunodeficiency virus (HIV) envelope glycoprotein 120-derived peptide P18-110 (RGPGRAFVTI) bound to the murine major histocompatibility complex (MHC) class I molecule, H-2Dd. This cDNA was then expressed in a bacterial vector, and protein, as inclusion bodies, was solubilized, refolded, and purified to homogeneity. Yield of the refolded material was from 10 to 50 mg per liter of bacterial culture, the protein was soluble at concentrations as high as 25 mg/ml, and it retained a high level of reactivity with an anti-V alpha 2 monoclonal antibody. This domain was monomeric both by size exclusion gel chromatography and by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Circular dichroism spectra indicated that the folded V alpha domain had secondary structure similar to that of single immunoglobulin or TCR domains, consisting largely of beta sheet. Conditions for crystallization were established, and at least two crystal geometries were observed: hexagonal bipyramids that failed to diffract beyond approximately 6 A, and orthorhombic crystals that diffracted to 2.5 A. The dimerization of the V alpha domain was investigated further by solution nuclear magnetic resonance spectroscopy, which indicated that dimeric and monomeric forms of the protein were about equally populated at a concentration of 1 mM. Thus, models of TCR-mediated T cell activation that invoke TCR dimerization must consider that some V alpha domains have little tendency to form homodimers or multimers.

scholarly journals Induction of Glutathione S-Transferase in Biofilms and Germinating Spores of Mucor hiemalis Strain EH5 from Cold Sulfidic Spring Waters

2007 ◽  
Vol 73 (8) ◽  
pp. 2697-2707 ◽  
Author(s):  
Enamul Hoque ◽  
Stephan Pflugmacher ◽  
Johannes Fritscher ◽  
Manfred Wolf
Keyword(s):  
High Performance ◽  
Laser Scanning ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Thiosulfate ◽  
Linear Increase ◽  
Size Exclusion ◽  
Mucor Hiemalis ◽  
Activity Measurements ◽  
Gst Activity

ABSTRACT The occurrence and activation of glutathione S-transferase (GST) and the GST activities in biofilms in cold sulfidic spring waters were compared to the occurrence and activation of GST and the GST activities of the aquatic fungal strains EH5 and EH7 of Mucor hiemalis isolated for the first time from such waters. Using fluorescently labeled polyclonal anti-GST antibodies and GST activity measurements, we demonstrated that a high level of GST occurred in situ in natural biofilms and pure cultures of strain EH5. Measurement of microsomal and cytosolic soluble GST activities using different xenobiotic substrates, including 1-chloro-2,4-dinitrobenzene (CDNB), 1,2-dichloro-4-nitrobenzene, 1,2-epoxy-3-(4-nitrophenoxy)propane, 1-iodo-2,4-dinitrobenzene, and fluorodifen, showed that the overall biotransforming abilities of biofilms were at least sixfold greater than that of strain EH5 alone. Increasing the level of sodium thiosulfate (STS) in the medium stimulated the microsomal and cytosolic GST activities with CDNB of strain EH5 about 44- and 94-fold, respectively, compared to the activities in the control. The induction of microsomal GST activity with fluorodifen by STS was strongly linear, but the initial strong linear increase in cytosolic GST activity with fluorodifen showed saturation-like effects at STS concentrations higher than approximately 1 mM. Using laser scanning confocal and conventional fluorescence microscopy, abundant fluorescently labeled GST proteins were identified in germinating sporangiospores of strain EH5 after activation by STS. High-performance size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of at least two main GSTs (∼27.8- and ∼25.6-kDa subunits) in the cytosol of EH5, whereas the major 27.8-kDa subunit was the only GST in microsomes. We suggest that differential cellular GST expression takes place in strain EH5 depending on spore and hyphal development. Our results may contribute to our understanding of induction of GST by sulfurous compounds, as well as to the immunofluorescence visualization of GST in aquatic fungus and fungus-bacterium biofilms.

Evaluation of EPS extraction protocols from anaerobic sludge for gel-based proteomic studies

2015 ◽  
Vol 72 (4) ◽  
pp. 535-542 ◽  
Author(s):  
J. A. Zorel ◽  
S. F. Aquino ◽  
A. L. Sanson ◽  
W. Castro-Borges ◽  
S. Q. Silva
Keyword(s):  
Extracellular Polymeric Substances ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Background Level ◽  
Molecular Size ◽  
Extraction Methods ◽  
Extraction Yield ◽  
Size Exclusion ◽  
Anaerobic Sludge ◽  
Distribution Profile

Despite the importance of anaerobic sludge extracellular polymeric substances (EPSs), their characterization is limited to information regarding their chemical classes and molecular size. This work explores the possibility of using proteomic techniques to study the proteins present in this matrix. Thus, this paper compares eight EPS extraction methods regarding extraction yield, protein/carbohydrate ratio, size distribution profile and suitability to sodium dodecyl sulfate–polyacrylamide gel electrophoresis analyses. Despite the differences found in quantification and size exclusion chromatography assays, the band profile found for all methods was very similar. Considering the band pattern, extraction time and background level, heating method followed by ammonium sulfate precipitation proved to be the most appropriate method for gel-based analyses of anaerobic sludge EPS proteins.

scholarly journals Purification of a Zinc Binding Protein from Xylem of Citrus jambhiri

2002 ◽  
Vol 127 (5) ◽  
pp. 718-723 ◽  
Author(s):  
Kathryn C. Taylor ◽  
Danielle R. Ellis ◽  
Luciano V. Paiva
Keyword(s):  
Total Protein ◽  
Proteinase Inhibitors ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sulfhydryl Group ◽  
Size Exclusion ◽  
Binding Studies ◽  
Citrus Jambhiri ◽  
Citrus Rootstock

Zinc in xylem and phloem of the citrus rootstock, rough lemon [Citrus jambhiri (L.)] was associated with a Zn-binding protein, designated citrus vascular Zn-binding protein (CVZBP). The apparent molecular mass of the CVZBP was 19.5 kDa after nondenaturing size exclusion chromatography and 21.8 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ion exchange chromatography demonstrated that CVZBP was anionic, requiring 0.43 n NaCl for elution from quaternary aminoethyl Sepharose. Antiserum to the protein cross-reacted more with total protein extracts from leaf midveins than with total protein from the rest of the leaf lamina, further suggesting a vascular location of the Zn-binding protein. Quantitative analysis indicated that ≈2 to 3 mol of Zn were associated with 1 mol of native protein. Binding studies with the partially purified CVZBP demonstrated a capacity to bind several divalent cations: Cd, Ni, Pb, and Zn. Reaction with Ellman's reagent suggested that the protein has significant sulfhydryl group content that may be involved in metal binding. N-terminal sequencing demonstrates identity with papaya latex trypsin inhibitor, sporamin, or other Kunitz soybean proteinase inhibitors.

scholarly journals The tumor suppressor p53 is bound to RNA by a stable covalent linkage.

1991 ◽  
Vol 11 (3) ◽  
pp. 1598-1606 ◽  
Author(s):  
A Samad ◽  
R B Carroll
Keyword(s):  
Tumor Suppressor ◽  
Tryptic Peptide ◽  
Polyacrylamide Gel Electrophoresis ◽  
Active Form ◽  
Sodium Dodecyl ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Biologically Active ◽  
Intact Protein ◽  
Tumor Suppressor P53

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.

scholarly journals Characterization of a Novel β-Galactosidase fromBifidobacterium adolescentis DSM 20083 Active towards Transgalactooligosaccharides

2000 ◽  
Vol 66 (4) ◽  
pp. 1379-1384 ◽  
Author(s):  
Katrien M. J. Van Laere ◽  
Tjakko Abee ◽  
Henk A. Schols ◽  
Gerrit Beldman ◽  
Alphons G. J. Voragen
Keyword(s):  
Size Exclusion Chromatography ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cell Extract ◽  
Degree Of Polymerization ◽  
Exchange Chromatography ◽  
Size Exclusion ◽  
Bifidobacterium Adolescentis ◽  
Exclusion Chromatography

ABSTRACT This paper reports on the effects of both reducing and nonreducing transgalactooligosaccharides (TOS) comprising 2 to 8 residues on the growth of Bifidobacterium adolescentis DSM 20083 and on the production of a novel β-galactosidase (β-Gal II). In cells grown on TOS, in addition to the lactose-degrading β-Gal (β-Gal I), another β-Gal (β-Gal II) was detected and it showed activity towards TOS but not towards lactose. β-Gal II activity was at least 20-fold higher when cells were grown on TOS than when cells were grown on galactose, glucose, and lactose. Subsequently, the enzyme was purified from the cell extract of TOS-grown B. adolescentis by anion-exchange chromatography, adsorption chromatography, and size-exclusion chromatography. β-Gal II has apparent molecular masses of 350 and 89 kDa as judged by size-exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, indicating that the enzyme is active in vivo as a tetramer. β-Gal II had an optimal activity at pH 6 and was not active below pH 5. Its optimum temperature was 35°C. The enzyme showed highestV max values towards galactooligosaccharides with a low degree of polymerization. This result is in agreement with the observation that during fermentation of TOS, the di- and trisaccharides were fermented first. β-Gal II was active towards β-galactosyl residues that were 1→4, 1→6, 1→3, and 1↔1 linked, signifying its role in the metabolism of galactooligosaccharides by B. adolescentis.

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