scholarly journals Decoding sequence-level information to predict membrane protein expression

2017 ◽  
Author(s):  
Shyam M. Saladi ◽  
Nauman Javed ◽  
Axel Müller ◽  
William M. Clemons
Keyword(s):  
Sequence Information ◽  
Biophysical Characterization ◽  
E Coli ◽  
Membrane Protein Expression ◽  
Improve Model ◽  
Wide Range ◽  
Probability Of Success ◽  
Level Information ◽  
Major Bottleneck

SummaryThe expression of integral membrane proteins (IMPs) remains a major bottleneck in the characterization of this important protein class. IMP expression levels are currently unpredictable, which renders the pursuit of IMPs for structural and biophysical characterization challenging and inefficient. Experimental evidence demonstrates that changes within the nucleotide or amino-acid sequence for a given IMP can dramatically affect expression; yet these observations have not resulted in generalizable approaches to improved expression. Here, we develop a data-driven statistical predictor named IMProve, that, using only sequence information, increases the likelihood of selecting an IMP that expresses in E. coli. The IMProve model, trained on experimental data, combines a set of sequence-derived features resulting in an IMProve score, where higher values have a higher probability of success. The model is rigorously validated against a variety of independent datasets that contain a wide range of experimental outcomes from various IMP expression trials. The results demonstrate that use of the model can more than double the number of successfully expressed targets at any experimental scale. IMProve can immediately be used to identify favorable targets for characterization.

Biophysical characterization of E. coli TolC interaction with the known blocker hexaamminecobalt

2017 ◽  
Vol 1861 (11) ◽  
pp. 2702-2709 ◽  
Author(s):  
A. Gilardi ◽  
S.P. Bhamidimarri ◽  
M. Brönstrup ◽  
U. Bilitewski ◽  
R.K.R. Marreddy ◽  
...  
Keyword(s):  
Biophysical Characterization ◽  
E Coli

scholarly journals Adsorption to soils and biochemical characterization of purified phytases

2019 ◽  
Author(s):  
Maria Marta Caffaro ◽  
Karina Beatriz Balestrasse ◽  
Gerardo Rubio
Keyword(s):  
Phytic Acid ◽  
Crop Production ◽  
Biochemical Characterization ◽  
Solid Phase ◽  
Organic P ◽  
E Coli ◽  
Nitrophenyl Phosphate ◽  
Wide Range ◽  
Temperature Ranges

Abstract. Four purified phytases isolated from Aspergillus niger and Escherichia coli were characterized biochemically and in terms of their adsorption to soils belonging to the Mollisol order. Three different organic P substrates were used to measure enzyme activity in a wide range of pH (2.3 to 9) and temperatures (−10° to 70 °C): p-nitrophenyl-phosphate, glyceraldehyde-3-phosphate and phytic acid. Phytases from A. niger showed a higher capacity to release P (36 to 50 % of P contained in the substrates, 44 to 62 μg P), than phytases from E. coli (24 to 36 %, 20 to 44 μg P). The amount of P released from organic P substrates by A. niger phytases followed the following range: p-nitrophenyl-phosphate > glyceraldehyde-3-phosphate > phytic acid whereas in E. coli phytases the order was p-nitrophenyl-phosphate/glyceraldehyde-3-phosphate > phytic acid. All phytases were active throughout the pH and temperature ranges for optimum crop production. The proportion of phytases found in the solid phase of the soil 60 minutes after addition was lower than that found in the liquid phase (23–34 % vs. 66–77 %). Obtained results are promising in terms of the use of phytases as a complement to P fertilization in agricultural settings and encourages further studies under field conditions.

scholarly journals Identification and Characterization of Two Klebsiella pneumoniaelpxL Lipid A Late Acyltransferases and Their Role in Virulence

2017 ◽  
Vol 85 (9) ◽  
Author(s):  
Grant Mills ◽  
Amy Dumigan ◽  
Timothy Kidd ◽  
Laura Hobley ◽  
José A. Bengoechea
Keyword(s):  
Lipid A ◽  
Galleria Mellonella ◽  
Inflammatory Responses ◽  
Two Component System ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Candidate Target ◽  
Tract Infections

ABSTRACT Klebsiella pneumoniae causes a wide range of infections, from urinary tract infections to pneumonia. The lipopolysaccharide is a virulence factor of this pathogen, although there are gaps in our understanding of its biosynthesis. Here we report on the characterization of K. pneumoniae lpxL, which encodes one of the enzymes responsible for the late secondary acylation of immature lipid A molecules. Analysis of the available K. pneumoniae genomes revealed that this pathogen's genome encodes two orthologues of Escherichia coli LpxL. Using genetic methods and mass spectrometry, we demonstrate that LpxL1 catalyzes the addition of laureate and LpxL2 catalyzes the addition of myristate. Both enzymes acylated E. coli lipid A, whereas only LpxL2 mediated K. pneumoniae lipid A acylation. We show that LpxL1 is negatively regulated by the two-component system PhoPQ. The lipid A produced by the lpxL2 mutant lacked the 2-hydroxymyristate, palmitate, and 4-aminoarabinose decorations found in the lipid A synthesized by the wild type. The lack of 2-hydroxymyristate was expected since LpxO modifies the myristate transferred by LpxL2 to the lipid A. The absence of the other two decorations is most likely caused by the downregulation of phoPQ and pmrAB expression. LpxL2-dependent lipid A acylation protects Klebsiella from polymyxins, mediates resistance to phagocytosis, limits the activation of inflammatory responses by macrophages, and is required for pathogen survival in the wax moth (Galleria mellonella). Our findings indicate that the LpxL2 contribution to virulence is dependent on LpxO-mediated hydroxylation of the LpxL2-transferred myristate. Our studies suggest that LpxL2 might be a candidate target in the development of anti-K. pneumoniae drugs.

scholarly journals Biophysical characterization of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2

2020 ◽  
Author(s):  
Talita Stelling de Araújo ◽  
Sandra M. N. Scapin ◽  
William de Andrade ◽  
Maira Fasciotti ◽  
Mariana T. Q. de Magalhães ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Lymphoblastic Leukemia ◽  
Biophysical Characterization ◽  
E Coli ◽  
Terminal Loop ◽  
Hydrolysis Of

AbstractThe hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. Here we present a comparative study two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2, performed by a comprehensive array of biophysical and biochemical approaches. We report the oligomeric landscape and conformational and dynamic plasticity of E. coli type 2 L-asparaginase (EcA2) present in two different formulations, and its relationship with L-aspartic acid, which is present in Aginasa, but not in Leuginase. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. The N-terminal loop of Leuginase, which is part of the active site is flexibly disordered, but gets ordered as in Aginasa in the presence os L-Asp, while L-Glu only does so to a limited extent. Ion-mobility spectrometry–mass spectrometry reveals two conformers for the monomeric EcA2, one of which can selectively bind to L-Asp and L-Glu. Aginasa has higher resistance to in vitro proteolysis than Leuginase, and this is directly related to the presence of L-Asp.

scholarly journals Construction and characterization of a recombinant mutant homolog of the CheW protein from Thermotoga petrophila RKU-1

2018 ◽  
Vol 64 (1) ◽  
pp. 53-60 ◽  
Author(s):  
D.V. Grishin ◽  
D.D. Zhdanov ◽  
Ju.A. Gladilina ◽  
V.S. Pokrovsky ◽  
O.V. Podobed ◽  
...  
Keyword(s):  
Heat Treatment ◽  
Broad Spectrum ◽  
Industrial Application ◽  
Intestinal Bacteria ◽  
Preliminary Heat Treatment ◽  
E Coli ◽  
Thermotoga Petrophila ◽  
Wide Range ◽  
High Expression Level

In the work a recombinant chemotaxis protein CheW from Thermotoga petrophila RKU-1 (TpeCheW) and its mutant homolog (TpeCheW-mut) were created. It was shown that, despite the low homology with CheW prototypes from intestinal bacteria, these proteins didn't cause metabolic overload and were well expressed by cells of E. coli laboratory strains. We have discovered a broad spectrum of industrial valuable properties of the TpeCheW-mut protein such as stability in a wide range of temperatures and pH, high expression level, solubility and possibility of the application of a simple low-stage purification methodology with the use of preliminary heat treatment. Possible directions of the scientific and industrial application of this protein were claimed.

scholarly journals Biochemical and Biophysical Characterization of Recombinant Human 3-Phosphoglycerate Dehydrogenase

2021 ◽  
Vol 22 (8) ◽  
pp. 4231
Author(s):  
Giulia Murtas ◽  
Giorgia Letizia Marcone ◽  
Alessio Peracchi ◽  
Erika Zangelmi ◽  
Loredano Pollegioni
Keyword(s):  
Cancer Progression ◽  
Forward Direction ◽  
Biochemical Properties ◽  
Reverse Direction ◽  
Phosphate Binding ◽  
Biophysical Characterization ◽  
E Coli ◽  
Phosphoglycerate Dehydrogenase ◽  
Serine Metabolism

The human enzyme D-3-phosphoglycerate dehydrogenase (hPHGDH) catalyzes the reversible dehydrogenation of 3-phosphoglycerate (3PG) into 3-phosphohydroxypyruvate (PHP) using the NAD+/NADH redox cofactor, the first step in the phosphorylated pathway producing L-serine. We focused on the full-length enzyme that was produced in fairly large amounts in E. coli cells; the effect of pH, temperature and ligands on hPHGDH activity was studied. The forward reaction was investigated on 3PG and alternative carboxylic acids by employing two coupled assays, both removing the product PHP; 3PG was by far the best substrate in the forward direction. Both PHP and α-ketoglutarate were efficiently reduced by hPHGDH and NADH in the reverse direction, indicating substrate competition under physiological conditions. Notably, neither PHP nor L-serine inhibited hPHGDH, nor did glycine and D-serine, the coagonists of NMDA receptors related to L-serine metabolism. The investigation of NADH and phosphate binding highlights the presence in solution of different conformations and/or oligomeric states of the enzyme. Elucidating the biochemical properties of hPHGDH will enable the identification of novel approaches to modulate L-serine levels and thus to reduce cancer progression and treat neurological disorders.

scholarly journals The development and characterization of an E. coli O25B bioconjugate vaccine

2021 ◽  
Author(s):  
Michael Kowarik ◽  
Michael Wetter ◽  
Micha A. Haeuptle ◽  
Martin Braun ◽  
Michael Steffen ◽  
...  
Keyword(s):  
Campylobacter Jejuni ◽  
Multidrug Resistant ◽  
Sequence Type ◽  
Detailed Characterization ◽  
E Coli ◽  
Wide Range ◽  
Physicochemical Methods ◽  
Tract Infections ◽  
Opening Up

AbstractExtraintestinal pathogenic Escherichia coli (ExPEC) cause a wide range of clinical diseases such as bacteremia and urinary tract infections. The increase of multidrug resistant ExPEC strains is becoming a major concern for the treatment of these infections and E. coli has been identified as a critical priority pathogen by the WHO. Therefore, the development of vaccines has become increasingly important, with the surface lipopolysaccharide constituting a promising vaccine target. This study presents genetic and structural analysis of clinical urine isolates from Switzerland belonging to the serotype O25. Approximately 75% of these isolates were shown to correspond to the substructure O25B only recently described in an emerging clone of E. coli sequence type 131. To address the high occurrence of O25B in clinical isolates, an O25B glycoconjugate vaccine was prepared using an E. coli glycosylation system. The O antigen cluster was integrated into the genome of E. coli W3110, thereby generating an E. coli strain able to synthesize the O25B polysaccharide on a carrier lipid. The polysaccharide was enzymatically conjugated to specific asparagine side chains of the carrier protein exotoxin A (EPA) of Pseudomonas aeruginosa by the PglB oligosaccharyltransferase from Campylobacter jejuni. Detailed characterization of the O25B-EPA conjugate by use of physicochemical methods including NMR and GC-MS confirmed the O25B polysaccharide structure in the conjugate, opening up the possibility to develop a multivalent E. coli conjugate vaccine containing O25B-EPA.

scholarly journals Oligoribonuclease Is Encoded by a Highly Conserved Gene in the 3′-5′ Exonuclease Superfamily

1998 ◽  
Vol 180 (10) ◽  
pp. 2779-2781 ◽  
Author(s):  
Xiaolan Zhang ◽  
Liuqin Zhu ◽  
Murray P. Deutscher
Keyword(s):  
Kanamycin Resistance ◽  
Open Reading Frame ◽  
Sequence Information ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli ◽  
Kanamycin Resistance Cassette ◽  
Wide Range ◽  
Conserved Gene ◽  
Cloned Gene

ABSTRACT Oligoribonuclease, a 3′-to-5′ exoribonuclease specific for small oligoribonucleotides, was purified to homogeneity from extracts ofEscherichia coli. The purified protein is an α2 dimer of 40 kDa. NH2-terminal sequence analysis of the protein identified the gene encoding oligoribonuclease as yjeR(o204a), a previously reported open reading frame located at 94 min on the E. coli chromosome. However, as a consequence of the sequence information, the translation start site of this open reading frame has been revised. Cloning of yjeRled to overexpression of oligoribonuclease activity, and interruption of the cloned gene with a kanamycin resistance cassette eliminated the overexpression. On the basis of these data, we propose thatyjeR be renamed orn. Orthologs of oligoribonuclease are present in a wide range of organisms, extending up to humans.

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