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.

scholarly journals Restoration of β-galactosidase to Escherichia coli M15. Complementation studies

1977 ◽  
Vol 165 (3) ◽  
pp. 417-423 ◽  
Author(s):  
Dobrivoje V. Marinkovic ◽  
Jelka N. Marinkovic
Keyword(s):  
Escherichia Coli ◽  
Sodium Dodecyl Sulphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Galactosidase Activity ◽  
Terminal Amino Acid ◽  
E Coli ◽  
Molecular Weights ◽  
Terminal Amino

Carboxymethylated β-galactosidase from Escherichia coli was dissociated at 100°C to form carboxymethylated fragments A and B. The mol.wts. of carboxymethylated fragments A and B were determined by gel filtration to be 64300 and 22400 respectively. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of carboxymethylated fragments A and B that had been pretreated with 2-mercaptoethanol and sodium dodecyl sulphate yielded mol.wts. of 64000 and 22100 respectively. Carboxymethylated fragments A and B had arginine as their C-terminal amino acid. When a crude extract of E. coli M15 was filtered through a column of Sepharose 6B, it was found that carboxymethylated fragment B could restore β-galactosidase activity when added to fractions having mol.wts. estimated to be 123000, 262000 and 506000. These fractions are referred to as ‘complementable fractions’. Similarly, it was found that carboxymethylated fragment A could restore enzyme activity to tractions having mol.wts. estimated to be 63000, 253000 and 506000. Estimates of the molecular weights of the β-galactosidase activity obtained by restoration with carboxymethylated fragments A and B were made by filtering the active enzyme through another column of Sepharose 6B. The enzyme obtained by complementation with carboxymethylated fragment B, i.e. the complemented enzyme, had mol.wt. 525000, and that obtained with carboxymethylated fragment A had mol.wts. of 525000, 646000 and 2000000. The latter finding suggests that multiple forms of complemented β-galactosidase can exist.

Isolation of Fasciola hepatica haemoglobin

Parasitology ◽  
1995 ◽  
Vol 111 (2) ◽  
pp. 209-215 ◽  
Author(s):  
S. McGonigle ◽  
J. P. Dalton
Keyword(s):  
Fasciola Hepatica ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequence Analysis ◽  
Terminal Amino Acid ◽  
Terminal Amino ◽  
Haemoglobin Molecule

SUMMARYA haemoprotein released in vitro by adult Fasciola hepatica was purified by gel filtration chromatography on Sephacryl S-200 and ion-exchange chromatography on DEAE-Sepharose. The molecule, with an apparent molecular weight of > 200 kDa, contains a haem group and has absorption spectra characteristics similar to haemoglobins. N-terminal amino acid sequence analysis revealed no similarity between the F. hepatica haemoglobin and other vertebrate or invertebrate haemoglobins. Antibodies to the haemoglobin molecule can be detected in the sera of F. hepatica-infected bovines as early as 1 week after infection.

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 Expression, biotinylation and purification of a biotin-domain peptide from the biotin carboxy carrier protein of Escherichia coli acetyl-CoA carboxylase

1994 ◽  
Vol 302 (3) ◽  
pp. 881-887 ◽  
Author(s):  
A Chapman-Smith ◽  
D L Turner ◽  
J E Cronan ◽  
T W Morris ◽  
J C Wallace
Keyword(s):  
Escherichia Coli ◽  
Exchange Chromatography ◽  
Carrier Protein ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
E Coli ◽  
Biotin Ligase ◽  
Domain Peptide

A protein segment consisting of the C-terminal 87 residues of the biotin carboxy carrier protein from Escherichia coli acetyl-CoA carboxylase was overexpressed in E. coli. The expressed biotin-domain peptide can be fully biotinylated by coexpression with a plasmid that overproduces E. coli biotin ligase. The extent of biotinylation was limited in vivo, but could be taken to completion in cell lysates on addition of ATP and biotin. We used the coexpression of biotin ligase and acceptor protein to label the biotin-domain peptide in vitro with [3H]biotin, which greatly facilitated development of a purification procedure. The apo (unbiotinylated) form of the protein was prepared by induction of biotin-domain expression in a strain lacking the biotin-ligase-overproduction plasmid. The apo domain could be separated from the biotinylated protein by ion-exchange chromatography or non-denaturing PAGE, and was converted into the biotinylated form of the peptide on addition of purified biotin ligase. The identify of the purified biotin-domain peptide was confirmed by N-terminal sequence analysis, amino acid analysis and m.s. The domain was readily produced and purified in sufficient quantities for n.m.r. structural analysis.

A fraction from Escherichia coli with anti-Aspergillus properties

2005 ◽  
Vol 54 (4) ◽  
pp. 375-379 ◽  
Author(s):  
V Yadav ◽  
R Mandhan ◽  
Rajesh Dabur ◽  
A K Chhillar ◽  
J Gupta ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Concentration ◽  
Exchange Chromatography ◽  
Active Molecule ◽  
Standard Drug ◽  
E Coli ◽  
Variable Activity ◽  
Sds Page ◽  
Haemolytic Assay

The products of various strains of Escherichia coli (BL21, DH5α, HB101 and XL Blue) were investigated for antimycotic properties using pathogenic isolates of Aspergillus. Co-culture experiments revealed that E. coli strains exhibited variable activity against Aspergillus fumigatus. The lysates prepared from DH5α, HB101 and XL Blue strains of E. coli showed inhibitory activity against A. fumigatus in the protein concentration range of 62.50 to 250.00 μg ml−1. The highest activity was seen in the lysate of BL21, which inhibited the growth of A. fumigatus and Aspergillus flavus completely at a concentration of 31.25 μg protein ml−1. The MIC of BL21 lysate against Aspergillus niger was found to be 62.50 μg ml−1. The in vitro toxicity of BL21 lysate was evaluated using a haemolytic assay. A BL21 lysate protein concentration of 1250.00 μg ml−1 was found to be nontoxic to human erythrocytes. The standard drug amphotericin B lysed 100 % of erythrocytes at a concentration of 37.50 μg ml−1. SDS-PAGE showed the presence of at least 15 major proteins in the lysate of BL21. Ion-exchange chromatography resolved the BL21 lysate into five fractions and fraction III was found to be endowed with anti-Aspergillus properties. The MIC of this fraction was found to be 3.90 μg ml−1. Further work on the purification of the active molecule and its characterization is in progress.

Kynurenine aminotransferase and glutamine transaminase K of Escherichia coli: identity with aspartate aminotransferase

2001 ◽  
Vol 360 (3) ◽  
pp. 617-623 ◽  
Author(s):  
Qian HAN ◽  
Jianmin FANG ◽  
Jianyong LI
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Aspartate Aminotransferase ◽  
Expression System ◽  
Xanthurenic Acid ◽  
Amino Acid Residues ◽  
Terminal Amino Acid ◽  
Kynurenine Aminotransferase ◽  
E Coli ◽  
Terminal Amino

The present study describes the isolation of a protein from Escherichia coli possessing kynurenine aminotransferase (KAT) activity and its identification as aspartate aminotransferase (AspAT). KAT catalyses the transamination of kynurenine and 3-hydroxykynurenine to kynurenic acid and xanthurenic acid respectively, and the enzyme activity can be easily detected in E. coli cells. Separation of the E. coli protein possessing KAT activity through various chromatographic steps led to the isolation of the enzyme. N-terminal sequencing of the purified protein determined its first 10 N-terminal amino acid residues, which were identical with those of the E. coli AspAT. Recombinant AspAT (R-AspAT), homologously expressed in an E. coli/pET22b expression system, was capable of catalysing the transamination of both l-kynurenine (Km = 3mM; Vmax = 7.9μmol·min−1·mg−1) and 3-hydroxy-dl-kynurenine (Km = 3.7mM; Vmax = 1.25μmol·min−1·mg−1) in the presence of pyruvate as an amino acceptor, and exhibited its maximum activity at temperatures between 50–60°C and at a pH of approx. 7.0. Like mammalian KATs, R-AspAT also displayed high glutamine transaminase K activity when l-phenylalanine was used as an amino donor (Km = 8mM; Vmax = 20.6μmol·min−1·mg−1). The exact match of the first ten N-terminal amino acid residues of the KAT-active protein with that of AspAT, in conjunction with the high KAT activity of R-AspAT, provides convincing evidence that the identity of the E. coli protein is AspAT.

Purification of Clostridium perfringens type C theta toxin

1973 ◽  
Vol 19 (8) ◽  
pp. 881-885 ◽  
Author(s):  
A. H. W. Hauschild ◽  
A. Lecroisey ◽  
J. E. Alouf
Keyword(s):  
Clostridium Perfringens ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Ammonium Sulfate Precipitation ◽  
Terminal Amino Acid ◽  
Single Protein ◽  
Dodecyl Sulfate ◽  
Type C ◽  
Terminal Amino ◽  
Anionic Polyacrylamide

Clostridium perfringens type C theta toxin was purified by ammonium sulfate precipitation, gel filtration, and ion-exchange chromatography. The purified toxin was free of alpha, beta, delta, and kappa toxins. Electrophoresis of the toxin in both the anionic polyacrylamide disc gel and in the polyacrylamide dodecyl sulfate gel yielded single protein bands. L-Lysine was determined as the sole N-terminal amino acid. The specific hemolytic activities of two purified preparations were 3.6 × 106 and 4.8 × 106 HU/mg N; the specific toxicities were 8.1 × 103 and 7.7 × 103 mouse MLD/mg N. The molecular weight determined by the polyacrylamide – dodecyl sulfate method was 74 000.

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.

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.

A ribosomal-boand aminopeptidase in Escherichia coli B: substrate specificity

1970 ◽  
Vol 48 (12) ◽  
pp. 1292-1296 ◽  
Author(s):  
A. T. Matheson ◽  
A. J. Dick ◽  
F. Rollin
Keyword(s):  
Escherichia Coli ◽  
Substrate Specificity ◽  
Peptide Bond ◽  
Terminal Amino Acid ◽  
Strong Activity ◽  
E Coli ◽  
Rate Of Hydrolysis ◽  
Terminal Amino ◽  
Hydrolysis Of ◽  
Escherichia Coli B

The substrate specificity of the ribosomal-bound aminopeptidase from Escherichia coli B has been studied using di-, tri-, and tetrapeptides. The enzyme shows strong activity to leucyl, methionyl, threonyl, and lysyl peptides. Of the other dipeptides tested considerable hydrolysis was observed only if the C-terminal amino acid was leucine or methionine. In a given series of peptides the rate of hydrolysis of the N-terminal peptide bond increased as the size of the peptide increased. Although leucyi dipeptides were hydroiyzed more rapidly than the corresponding methionyl dipeptide the reverse was true with the tripeptides tested. No carboxypeptidase activity was observed and peptides containing D-amino acids were not hydroiyzed. The substrate specificity of the aminopeptidase was compared with the known N-terminal sequences of E. coli proteins to determine whether the enzyme may be involved in the removal of N-formylmethionyl from newly synthesized polypeptides.

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