scholarly journals Thioredoxin from Bacillus acidocaldarius: characterization, high-level expression in Escherichia coli and molecular modelling

1997 ◽  
Vol 328 (1) ◽  
pp. 277-285 ◽  
Author(s):  
Simonetta BARTOLUCCI ◽  
Annamaria GUAGLIARDI ◽  
Emilia PEDONE ◽  
Donatella DE PASCALE ◽  
Raffaele CANNIO ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Modelling ◽  
Current Knowledge ◽  
Ion Pairs ◽  
Gel Filtration ◽  
Bovine Insulin ◽  
Exchange Chromatography ◽  
Three Dimensional Model ◽  
Scanning Calorimetry ◽  
E Coli

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx) was purified to homogeneity by anion-exchange chromatography and gel-filtration chromatography, based on its ability to catalyse the dithiothreitol-dependent reduction of bovine insulin disulphides. The protein has a molecular mass of 11577 Da, determined by electrospray mass spectrometry, a pI of 4.2, and its primary structure was obtained by automated Edman degradation after cleavage with trypsin and cyanogen bromide. The sequences of known bacterial Trxs were aligned at the active site: BacTrx has an identity ranging from 45 to 53% with all sequences except that of the unusual Anabaena strain 7120 Trx (37% identity). The gene coding for BacTrx was isolated by a strategy based on PCR gene amplification and cloned in a plasmid downstream of a lac-derived promoter sequence; the recombinant clone was used as the expression vector for Escherichia coli. The expression was optimized by varying both the time of cell growth and the time of exposure to the inducer isopropyl β-D-thiogalactoside; expressed BacTrx represents approx. 5% of the total cytosolic protein. CD spectra and differential scanning calorimetry measurements demonstrated that BacTrx is endowed with a higher conformational heat stability than the Trx from E. coli. Nanogravimetry experiments showed a lower content of bound water in BacTrx than in E. coli Trx, and a transition temperature approx. 10 °C higher for BacTrx. The three-dimensional model of the oxidized form of BacTrx was constructed by a comparative molecular modelling technique, using E. coli Trx and Anabaena strain 7120 Trx as reference proteins. Increased networks of ion-pairs and shorter loops emerged as major features of the BacTrx structure compared with those of the template proteins. The findings are discussed in the light of the current knowledge about molecular determinants of protein stability.

Production of PEGylated GCSF from Non-classical Inclusion Bodies Expressed in Escherichia coli

2021 ◽  
Author(s):  
Nguyen Thi My Trinh ◽  
Tran Linh Thuoc ◽  
Dang Thi Phuong Thao
Keyword(s):  
Escherichia Coli ◽  
Inclusion Bodies ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Insoluble Fraction ◽  
Exchange Chromatography ◽  
Native Page ◽  
E Coli ◽  
Sds Page ◽  
Stimulating Factor

Background: The recombinant human granulocyte colony stimulating factor con-jugated with polyethylene glycol (PEGylated GCSF) has currently been used as an efficient drug for the treatment of neutropenia caused by chemotherapy due to its long circulating half-life. Previous studies showed that Granulocyte Colony Stimula-ting Factor (GCSF) could be expressed as non-classical Inclusion Bodies (ncIBs), which contained likely correctly folded GCSF inside at low temperature. Therefore, in this study, a simple process was developed to produce PEGylated GCSF from ncIBs. Methods: BL21 (DE3)/pET-GCSF cells were cultured in the LiFlus GX 1.5 L bioreactor and the expression of GCSF was induced by adding 0.5 mM IPTG. After 24 hr of fermentation, cells were collected, resuspended, and disrupted. The insoluble fraction was obtained from cell lysates and dissolved in 0.1% N-lauroylsarcosine solution. The presence and structure of dissolved GCSF were verified using SDS-PAGE, Native-PAGE, and RP-HPLC analyses. The dissolved GCSF was directly used for the con-jugation with 5 kDa PEG. The PEGylated GCSF was purified using two purification steps, including anion exchange chromatography and gel filtration chromatography. Results: PEGylated GCSF was obtained with high purity (~97%) and was finally demonstrated as a form containing one GCSF molecule and one 5 kDa PEG molecule (monoPEG-GCSF). Conclusion: These results clearly indicate that the process developed in this study might be a potential and practical approach to produce PEGylated GCSF from ncIBs expressed in Escherichia coli (E. coli).

scholarly journals Production of the HBc Protein from Different HBV Genotypes in E. coli. Use of Reassociated HBc VLPs for Packaging of ss- and dsRNA

2021 ◽  
Vol 9 (2) ◽  
pp. 283
Author(s):  
Ivars Petrovskis ◽  
Ilva Lieknina ◽  
Andris Dislers ◽  
Juris Jansons ◽  
Janis Bogans ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
E Coli ◽  
The Core ◽  
Hbv Genotypes ◽  
Core Proteins ◽  
B Virus

The core proteins (HBc) of the hepatitis B virus (HBV) genotypes A, B, C, D, E, F, and G were cloned and expressed in Escherichia coli (E. coli), and HBc-formed virus-like particles (VLPs) were purified with ammonium sulfate precipitation, gel filtration, and ion exchange chromatography (IEX). The best VLP yield was found for the HBc of the HBV genotypes D and G. For the HBc of the HBV genotypes D, F, and G, the possibility of dissociation and reassociation maintaining the native HBc structure was demonstrated. Single-stranded (ss) and double-stranded (ds) ribonucleic acid (RNA) was successfully packed into HBc VLPs for the HBV genotypes D and G.

scholarly journals A cyanogen bromide fragment of β-galactosidase from Escherichia coli with α-donor activity in complementation of the enzyme from mutant M15

1976 ◽  
Vol 155 (2) ◽  
pp. 209-216 ◽  
Author(s):  
D V. Marinkovic ◽  
J N. Marinkovic
Keyword(s):  
Escherichia Coli ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Terminal Amino Acid ◽  
E Coli ◽  
Molecular Weights ◽  
Terminal Amino ◽  
Cyanogen Bromide Fragment ◽  
Donor Activity

Aminoethylated β-galactosidase from Escherichia coli was cleaved by CNBr. The fragment C4a was purified by gel filtration and ion-exchange chromatography. The molecular weight of the fragment C4a was determined to be 9000 +/- 600. The N-terminal amino acid was found to be isoleucine. Qualitative examination of homogeneity was carried out by disc-gel electrophoresis. The fragment C4a was shown to be active as an α donor in complementation of β-galactosidase activity in vitro with E. coli mutant M15, which has a deletion in the α region of the z gene. The molecular weights of complementable fractions from mutant M15 were found to be 123 000 +/- 2500 and 507 000 +/- 11 000, and of the complemented enzyme 522 500 +/- 11 400.

Thermotoga neapolitana Homotetrameric Xylose Isomerase Is Expressed as a Catalytically Active and Thermostable Dimer in Escherichia coli

1998 ◽  
Vol 64 (7) ◽  
pp. 2357-2360 ◽  
Author(s):  
J. Michael Hess ◽  
Vladimir Tchernajenko ◽  
Claire Vieille ◽  
J. Gregory Zeikus ◽  
Robert M. Kelly
Keyword(s):  
Escherichia Coli ◽  
Differential Scanning Calorimetry ◽  
Gel Filtration ◽  
Xylose Isomerase ◽  
Thermotoga Neapolitana ◽  
Thermal Transitions ◽  
Scanning Calorimetry ◽  
Catalytically Active ◽  
The Stability ◽  
Tetrameric Form

ABSTRACT The xylA gene from Thermotoga neapolitana5068 was expressed in Escherichia coli. Gel filtration chromatography showed that the recombinant enzyme was both a homodimer and a homotetramer, with the dimer being the more abundant form. The purified native enzyme, however, has been shown to be exclusively tetrameric. The two enzyme forms had comparable stabilities when they were thermoinactivated at 95°C. Differential scanning calorimetry revealed thermal transitions at 99 and 109.5°C for both forms, with an additional shoulder at 91°C for the tetramer. These results suggest that the association of the subunits into the tetrameric form may have little impact on the stability and biocatalytic properties of the enzyme.

scholarly journals Natural Genetic Transformation of Clinical Isolates of Escherichia coli in Urine and Water

2002 ◽  
Vol 68 (1) ◽  
pp. 440-443 ◽  
Author(s):  
Markus Woegerbauer ◽  
Bernard Jenni ◽  
Florian Thalhammer ◽  
Wolfgang Graninger ◽  
Heinz Burgmann
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Current Knowledge ◽  
Antibiotic Resistance Genes ◽  
Aquatic Environments ◽  
Wild Type ◽  
E Coli ◽  
Naturally Occurring ◽  
Natural Genetic Transformation ◽  
Type Strains

ABSTRACT Transfer of plasmid-borne antibiotic resistance genes in Escherichia coli wild-type strains is possible by transformation under naturally occurring conditions in oligotrophic, aquatic environments containing physiologic concentrations of calcium. In contrast, transformation is suppressed in nitrogen-rich body fluids like urine, a common habitat of uropathogenic strains. Current knowledge indicates that transformation of these E. coli wild-type strains is of no relevance for the acquisition of resistance in this clinically important environment.

scholarly journals Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1968 ◽  
Vol 106 (2) ◽  
pp. 531-541 ◽  
Author(s):  
P T Grant ◽  
K. B. M. Reid
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Bovine Insulin ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Performic Acid ◽  
Amino Acid Residues ◽  
Islet Tissue ◽  
A Chain

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11·5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.

Purification and characterization of membrane-bound fumarate reductase from anaerobically grown Escherichia coli

1979 ◽  
Vol 57 (6) ◽  
pp. 813-821 ◽  
Author(s):  
Peter Dickie ◽  
Joel H. Weiner
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Sodium Cholate ◽  
Defined Medium ◽  
Exchange Chromatography ◽  
Fumarate Reductase ◽  
Molar Ratios ◽  
Membrane Bound

Fumarate reductase has been purified 100-fold to 95% homogeneity from the cytoplasmic membrane of Escherichia coli, grown anaerobically on a defined medium containing glycerol plus fumarate. Optimal solubilization of total membrane protein and fumarate reductase activity occurred with nonionic detergents having a hydrophobic–lipophilic balance (HLB) number near 13 and we routinely solubilized the enzyme with Triton X-100 (HLB number = 13.5). Membrane enzyme extracts were fractionated by hydrophobic-exchange chromatography on phenyl Sepharose CL-4B to yield purified enzyme. The enzyme, whether membrane bound, in Triton extracts, or purified, had an apparent Km near 0.42 mM. Two peptides with molecular weights of 70 000 and 24 000, present in 1:1 molar ratios, were identified by sodium dodecyl sulfate polyacrylamide slab-gel electrophoresis to coincide with enzyme activity. A minimal native molecular weight of 100 000 was calculated for fumarate reductase by Sephacryl S-200 gel filtration in the presence of sodium cholate. This would indicate that the enzyme is a dimer. The purified enzyme has low, but measurable, succinate dehydrogenase activity.

scholarly journals Impacts of Hematite Nanoparticle Exposure on Biomechanical, Adhesive, and Surface Electrical Properties of Escherichia coli Cells

2012 ◽  
Vol 78 (11) ◽  
pp. 3905-3915 ◽  
Author(s):  
Wen Zhang ◽  
Joseph Hughes ◽  
Yongsheng Chen
Keyword(s):  
Escherichia Coli ◽  
Electrical Properties ◽  
Current Knowledge ◽  
Statistical Significance ◽  
Spring Constant ◽  
Bacterial Cells ◽  
Kelvin Probe ◽  
Content Type ◽  
Force Microscopy ◽  
E Coli

ABSTRACTDespite a wealth of studies examining the toxicity of engineered nanomaterials, current knowledge on their cytotoxic mechanisms (particularly from a physical perspective) remains limited. In this work, we imaged and quantitatively characterized the biomechanical (hardness and elasticity), adhesive, and surface electrical properties ofEscherichia colicells with and without exposure to hematite nanoparticles (NPs) in an effort to advance our understanding of the cytotoxic impacts of nanomaterials. Both scanning electron microscopy (SEM) and atomic force microscopy (AFM) showed thatE. colicells had noticeable deformation with hematite treatment for 45 min with a statistical significance. The hematite-treated cells became significantly harder or stiffer than untreated ones, as evidenced by indentation and spring constant measurements. The average indentation of the hematite-treatedE. colicells was 120 nm, which is significantly lower (P< 0.01) than that of the untreated cells (approximately 400 nm). The spring constant of hematite-treatedE. colicells (0.28 ± 0.11 nN/nm) was about 20 times higher than that of untreated ones (0.01 ± 0.01 nN/nm). The zeta potential ofE. colicells, measured by dynamic light scattering (DLS), was shown to shift from −4 ± 2 mV to −27 ± 8 mV with progressive surface adsorption of hematite NPs, a finding which is consistent with the local surface potential measured by Kelvin probe force microscopy (KPFM). Overall, the reported findings quantitatively revealed the adverse impacts of nanomaterial exposure on physical properties of bacterial cells and should provide insight into the toxicity mechanisms of nanomaterials.

scholarly journals Purification and characterization of two fructose diphosphate aldolases from Escherichia coli (Crookes' strain)

1973 ◽  
Vol 131 (4) ◽  
pp. 833-841 ◽  
Author(s):  
Donald Stribling ◽  
Richard N. Perham
Keyword(s):  
Escherichia Coli ◽  
Molecular Weight ◽  
Metal Ions ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Class Ii ◽  
Class I ◽  
E Coli ◽  
Km Value ◽  
Bivalent Metal Ions

Two fructose diphosphate aldolases (EC 4.1.2.13) were detected in extracts of Escherichia coli (Crookes' strain) grown on pyruvate or lactate. The two enzymes can be resolved by chromatography on DEAE-cellulose at pH7.5, or by gel filtration on Sephadex G-200, and both have been obtained in a pure state. One is a typical bacterial aldolase (class II) in that it is strongly inhibited by metal-chelating agents and is reactivated by bivalent metal ions, e.g. Ca2+, Zn2+. It is a dimer with a molecular weight of approx. 70000, and the Km value for fructose diphosphate is about 0.85mm. The other aldolase is not dependent on metal ions for its activity, but is inhibited by reduction with NaBH4 in the presence of substrate. The Km value for fructose diphosphate is about 20μm (although the Lineweaver–Burk plot is not linear) and the enzyme is probably a tetramer with molecular weight approx. 140000. It has been crystallized. On the basis of these properties it is tentatively assigned to class I. The appearance of a class I aldolase in bacteria was unexpected, and its synthesis in E. coli is apparently favoured by conditions of gluconeogenesis. Only aldolase of class II was found in E. coli that had been grown on glucose. The significance of these results for the evolution of fructose diphosphate aldolases is briefly discussed.

scholarly journals Novel Translation Initiation Regulation Mechanism in Escherichia coli ptrB Mediated by a 5′-Terminal AUG

2017 ◽  
Vol 199 (14) ◽  
Author(s):  
Heather J. Beck ◽  
Gary R. Janssen
Keyword(s):  
Escherichia Coli ◽  
Translation Initiation ◽  
Translational Regulation ◽  
Current Knowledge ◽  
Upstream Open Reading Frame ◽  
Model Organisms ◽  
Regulation Mechanism ◽  
Rna Regulation ◽  
Content Type ◽  
E Coli

ABSTRACT Alternative translation initiation mechanisms, distinct from the Shine-Dalgarno (SD) sequence-dependent mechanism, are more prevalent in bacteria than once anticipated. Translation of Escherichia coli ptrB instead requires an AUG triplet at the 5′ terminus of its mRNA. The 5′-terminal AUG (5′-uAUG) acts as a ribosomal recognition signal to attract ribosomes to the ptrB mRNA rather than functioning as an initiation codon to support translation of an upstream open reading frame. ptrB expression exhibits a stronger dependence on the 5′-uAUG than the predicted SD sequence; however, strengthening the predicted ptrB SD sequence relieves the necessity for the 5′-uAUG. Additional sequences within the ptrB 5′ untranslated region (5′-UTR) work cumulatively with the 5′-uAUG to control expression of the downstream ptrB coding sequence (CDS), thereby compensating for the weak SD sequence. Replacement of 5′-UTRs from other mRNAs with the ptrB 5′-UTR sequence showed a similar dependence on the 5′-uAUG for CDS expression, suggesting that the regulatory features contained within the ptrB 5′-UTR are sufficient to control the expression of other E. coli CDSs. Demonstration that the 5′-uAUG present on the ptrB leader mRNA is involved in ribosome binding and expression of the downstream ptrB CDS revealed a novel form of translational regulation. Due to the abundance of AUG triplets at the 5′ termini of E. coli mRNAs and the ability of ptrB 5′-UTR regulation to function independently of gene context, the regulatory effects of 5′-uAUGs on downstream CDSs may be widespread throughout the E. coli genome. IMPORTANCE As the field of synthetic biology continues to grow, a complete understanding of basic biological principles will be necessary. The increasing complexity of the synthetic systems highlights the gaps in our current knowledge of RNA regulation. This study demonstrates that there are novel ways to regulate canonical Shine-Dalgarno-led mRNAs in Escherichia coli, illustrating that our understanding of the fundamental processes of translation and RNA regulation is still incomplete. Even for E. coli, one of the most-studied model organisms, genes with translation initiation mechanisms that do not fit the canonical Shine-Dalgarno sequence paradigm are being revealed. Uncovering diverse mechanisms that control translational expression will allow synthetic biologists to finely tune protein production of desired gene products.

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