scholarly journals Characterization of human interleukin 2 derived from Escherichia coli

1985 ◽  
Vol 229 (2) ◽  
pp. 429-439 ◽  
Author(s):  
S M Liang ◽  
B Allet ◽  
K Rose ◽  
M Hirschi ◽  
C M Liang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Dna Sequence ◽  
Protein A ◽  
Interleukin 2 ◽  
Coding Region ◽  
Disulphide Bridge ◽  
E Coli ◽  
Escherichia Coli Cells

Interleukin 2 isolated from Escherichia coli cells expressing the human interleukin gene has been characterized. The observed properties of the protein have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural human interleukin 2. The purified E. coli-derived interleukin 2 is a monomeric protein of Mr 15 000 with a sedimentation velocity of 1.86S. The amino acid composition of the protein and isoelectric point (7.7) are consistent with that part of the translated DNA sequence of the gene corresponding to the mature protein. A single disulphide bridge was identified between Cys-58 and Cys-105. C.d. suggested that interleukin 2 is predominantly alpha-helical in secondary structure. The E. coli-derived protein differed from natural interleukin 2 in the presence of N-terminal methionine and also in the absence of a carbohydrate moiety. Removal of the coding region for the first three amino acids of the natural interleukin 2 protein sequence (Ala-Pro-Thr) by site-specific mutagenesis resulted in a protein with N-terminal serine. The possibility that the specificity of the E. coli ribosomal methionine aminopeptidase may not recognize the sequence NH2-Met-Xaa-Pro is discussed (where Xaa is any amino acid residue).

scholarly journals Purification and characterization of a diptericin homologue from Sarcophaga peregrina (flesh fly)

1992 ◽  
Vol 287 (2) ◽  
pp. 573-578 ◽  
Author(s):  
M Ishikawa ◽  
T Kubo ◽  
S Natori
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Bactericidal Activity ◽  
Fat Body ◽  
Amino Acid Sequencing ◽  
Purification And Characterization ◽  
E Coli ◽  
Escherichia Coli Cells

A protein with a molecular mass of 8 kDa was found to be synthesized specifically when the fat-body from injured Sarcophaga peregrina larvae was cultured in vitro. This protein was purified from the haemolymph of the injured larvae to near-homogeneity. Partial amino acid sequencing revealed that this protein is a diptericin homologue. It showed bactericidal activity on growing, but not resting Escherichia coli cells. E. coli cells become elongated on treatment with this protein.

scholarly journals Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

1996 ◽  
Vol 314 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Johanneke L. H. BUSCH ◽  
Jacques L. J. BRETON ◽  
Barry M. BARTLETT ◽  
Richard JAMES ◽  
E. Claude HATCHIKIAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Magnetic Circular Dichroism ◽  
Wild Type ◽  
E Coli ◽  
Excess Iron ◽  
Expression In Escherichia Coli ◽  
Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

scholarly journals Invasion Activity of a Mycobacterium tuberculosis Peptide Presented by the Escherichia coli AIDA Autotransporter

2002 ◽  
Vol 70 (12) ◽  
pp. 6846-6852 ◽  
Author(s):  
Nicola Casali ◽  
Marc Konieczny ◽  
M. Alexander Schmidt ◽  
Lee W. Riley
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Amino Acid ◽  
Hela Cells ◽  
Protein A ◽  
Functional Domain ◽  
E Coli ◽  
Amino Acid Region ◽  
Acid Fragment ◽  
Rho Family

ABSTRACT The mce1A gene of Mycobacterium tuberculosis was initially identified by its ability to promote uptake of Escherichia coli into HeLa cells. It was subsequently shown that this activity was confined to a 58-amino-acid region of the protein. A 72-amino-acid fragment (InvX) incorporating this active peptide was expressed in E. coli as a fusion to the AIDA (adhesin involved in diffuse adherence) autotransporter translocator, and its stable expression on the surface of the bacterium was demonstrated. Recombinant E. coli expressing InvX-AIDA showed extensive association with HeLa cells, and InvX was shown to be sufficient for internalization. Uptake was found to be both microtubule and microfilament dependent and required the Rho family of GTPases. Thus, the E. coli AIDA system facilitated both the qualitative and quantitative analysis of the functional domain of a heterologous protein.

Characterization of two plasmid-encoded cefotaximases found in clinical Escherichia coli isolates: CTX-M-65 and a novel enzyme, CTX-M-87

2009 ◽  
Vol 58 (6) ◽  
pp. 811-815 ◽  
Author(s):  
Jun Yin ◽  
Jun Cheng ◽  
Zhen Sun ◽  
Ying Ye ◽  
Yu-Feng Gao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Hydrolytic Activity ◽  
E Coli ◽  
High Affinity ◽  
Extended Spectrum ◽  
M 14

Three clinical strains of Escherichia coli (p168, p517 and p667) were collected in 2006 from three hospitals in Anhui Province (China). PCR and DNA sequencing revealed that E. coli p168 carried a novel extended-spectrum β-lactamase (ESBL), which was designated CTX-M-87. The extended-spectrum β-lactamase which was carried by E. coli p517 and E. coli p667 was previously named CTX-M-65. The deduced amino acid sequence of CTX-M-87, with pI 9.1, differed from that of CTX-M-14 by the substitutions Ala77→Val and Pro167→Leu. Like CTX-M-14, CTX-M-87 had a more potent hydrolytic activity against cefotaxime than against ceftazidime and had high affinity for cefuroxime and cefotaxime. These data show that mutations at position 167 in CTX-M do not always affect catalytic activity and substrate preference.

scholarly journals Molecular characterization of the Acremonium chrysogenum cefG gene product: the native deacetylcephalosporin C acetyltransferase is not processed into subunits

1999 ◽  
Vol 337 (3) ◽  
pp. 379-385 ◽  
Author(s):  
Javier VELASCO ◽  
Santiago GUTIERREZ ◽  
Sonia CAMPOY ◽  
Juan F. MARTIN
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Protein Band ◽  
Immunoaffinity Chromatography ◽  
Enzymic Activity ◽  
Acremonium Chrysogenum ◽  
Wild Type ◽  
E Coli ◽  
Protein Levels

Constructions starting at each of the three in-frame ATG codons of the Acremonium chrysogenum cefG gene (Met1, Met46 and Met60) were expressed in Escherichia coli, obtaining proteins of 49, 44 and 43 kDa, respectively. All three proteins showed deacetylcephalosporin C (DAC) acetyltransferase activity. The native A. chrysogenum DAC acetyltransferase was purified to electrophoretic homogeneity by immunoaffinity chromatography. It showed a molecular mass of 50 kDa by filtration in calibrated Sephadex G-75 SF or Superose 12 (FPLC) columns. The N-terminal end of the pure DAC acetyltransferase was Met-Leu-Pro-Ser-Ala-Gln-Val-Ala-Arg-Leu, which matched perfectly the deduced amino acid sequence starting at Met1. The putative α- and β-subunits of DAC acetyltransferase were also obtained in E. coli but showed no enzymic activity either separately or in combination. Immunoblotting (Western) analysis revealed that the 50 kDa DAC acetyltransferase showed high protein levels in A. chrysogenum cultures at 72 and 96 h and decreased sharply thereafter, but in all cases no detectable processing of the enzyme into subunits was found. Three different A. chrysogenum strains (including the wild-type Brotzu strain and two high-cephalosporin-producing mutants) showed the same unprocessed 50 kDa DAC acetyltransferase. The non-producer mutant ATCC 20371 showed no DAC acetyltransferase protein band but formed a normal transcript of 1.4 kb.

scholarly journals Characterization of a Group of Anaerobically Induced, fnr-Dependent Genes of Salmonella typhimurium

1999 ◽  
Vol 181 (19) ◽  
pp. 6092-6097 ◽  
Author(s):  
Yan Wei ◽  
Charles G. Miller
Keyword(s):  
Escherichia Coli ◽  
Salmonella Typhimurium ◽  
Dna Sequence ◽  
Abc Transporter ◽  
E Coli

ABSTRACT We have previously reported the isolation of a group of anaerobically regulated, fnr-dependent lacfusions in Salmonella typhimurium and have grouped theseoxd genes into classes based on map position. In order to identify these genes, we have replaced the original Mud-lacfusion in a member of each oxd class with the much smaller Mud-cam element, cloned the fusion, and determined DNA sequence sufficient to define the oxd gene. Several of the fusions correspond to previously known genes from S. typhimurium or Escherichia coli: oxd-4 = cbiA and oxd-11 = cbiK, oxd-5 = hybB, oxd-7 = dcuB, oxd-8 = moaB, oxd-12 = dmsA, and oxd-14 = napB (aeg-46.5). Two other fusions correspond to previously unknown loci: oxd-2 encodes an acetate/propionate kinase, and oxd-6 encodes a putative ABC transporter present in S. typhimurium but not in E. coli.

Nucleotide sequence of the thioredoxin gene from Escherichia coli

1984 ◽  
Vol 4 (11) ◽  
pp. 917-923 ◽  
Author(s):  
Jan-Olov Höög ◽  
Hedvig von Bahr-Lindström ◽  
Staffan Josephson ◽  
Betty J. Wallace ◽  
Sidney R. Kushner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Restriction Enzyme ◽  
Predicted Amino Acid Sequence ◽  
Coding Region ◽  
E Coli ◽  
Ribosome Binding ◽  
Sequence Method

The nucleotide sequence of the thioredoxin gene from Escherichia coli was determined. The structural gene was identified on a cloned 3-kb PvuII Iragment by hybridization with a synthetic oligodeoxyribonucleotide corresponding to a part of the amino acid sequence of thioredoxin. Restriction-enzyme fragments were used as templates in the dideoxy sequence method, directly and after subcloning into M13mp8. A segment of 450 nucleotides was determined using both strands7 alternatively, without extensive overlaps. The sequence contains the thioredoxin coding region, a potential ribosome-binding site, and a putative promotor region. The predicted amino acid sequence differs by two inversions from the previously given thioredoxin sequence. The revised sequence is presented and the results further show that thioredoxins from E. coli B and K12 are identical.

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