Engineering upstream transcriptional and translational signals of Bordetella pertussis serotype 2 fimbrial subunit protein for efficient expression in Escherichia coli: in vitro autoassembly of the expressed product into filamentous structures

ABSTRACT We constructed hybrid Bordetella pertussis-Escherichia coli RNA polymerases and compared productive interactions between transcription activators and cognate RNA polymerase subunits in an in vitro transcription system. Virulence-associated genes of B. pertussis, in the presence of their activator BvgA, are transcribed by all variants of hybrid RNA polymerases, whereas transcription at the E. coli lacpromoter regulated by the cyclic AMP-catabolite gene activator protein has an absolute requirement for the E. coli α subunit. This suggests that activator contact sites involve a high degree of selectivity.

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High level of soluble expression in Escherichia coli and characterisation of the CyaA pore-forming fragment from a Bordetella pertussis Thai clinical isolate

Archives of Microbiology ◽

10.1007/s00203-007-0302-1 ◽

2007 ◽

Vol 189 (2) ◽

pp. 169-174 ◽

Cited By ~ 13

Author(s):

Busaba Powthongchin ◽

Chanan Angsuthanasombat

Keyword(s):

Escherichia Coli ◽

Clinical Isolate ◽

Bordetella Pertussis ◽

Soluble Expression ◽

Expression In Escherichia Coli ◽

High Level

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Recombinant mouse prolactin: expression in Escherichia coli, purification and biological activity

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0080165 ◽

1992 ◽

Vol 8 (2) ◽

pp. 165-172 ◽

Cited By ~ 8

Author(s):

M. Yamamoto ◽

T. Harigaya ◽

T. Ichikawa ◽

K. Hoshino ◽

K. Nakashima

Keyword(s):

Escherichia Coli ◽

Biological Activity ◽

Amino Acid ◽

Multicopy Plasmid ◽

High Level Expression ◽

E Coli ◽

Expression In Escherichia Coli ◽

Oligonucleotide Duplex ◽

Efficient Expression ◽

High Level

ABSTRACT Transformation of Escherichia coli cells with a recombinant plasmid containing modified mouse prolactin (mPRL) cDNA and a pKK223-3 vector resulted in efficient expression of mPRL protein. Cloned mPRL cDNA was modified by removing the 5′ non-translating sequence as well as the sequence which encoded the signal peptide of preprolactin for recombination. In addition, approximately 100 nucleotides of the 5′-terminal region of the cDNA, which include the ATG initiation codon and the following 31 codons of mature mPRL, were replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen to be rich in AT without changing the amino acid sequence of the protein. The modified cDNA was finally inserted into the multicopy plasmid, pUC19, before high-level expression of mPRL in E. coli cells was obtained. Western blotting analysis of total protein from transformed E. coli cells showed that both 23 and 16kDa peptides were recognized by specific mPRL antisera. The purified and refolded 23 kDa protein exhibited a growth-stimulating effect on rat Nb 2 Node lymphoma cells, and was very similar to that of natural pituitary PRL.

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Nitazoxanide Inhibits Biofilm Production and Hemagglutination by Enteroaggregative Escherichia coli Strains by Blocking Assembly of AafA Fimbriae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01279-09 ◽

2010 ◽

Vol 54 (4) ◽

pp. 1526-1533 ◽

Cited By ~ 26

Author(s):

Eliah R. Shamir ◽

Michelle Warthan ◽

Sareena P. Brown ◽

James P. Nataro ◽

Richard L. Guerrant ◽

...

Keyword(s):

Escherichia Coli ◽

Antimicrobial Agents ◽

Luria Bertani ◽

Type I ◽

Reverse Transcription Pcr ◽

Fimbrial Subunit ◽

Biofilm Production ◽

Type I Fimbriae ◽

Fimbrial Adhesins

ABSTRACT Enteroaggregative Escherichia coli (EAEC) strains have emerged as common causes of persistent diarrhea and malnutrition among children and HIV-infected persons. During infection, EAEC typically adheres to the intestinal mucosa via fimbrial adhesins, which results in a characteristic aggregative pattern. In the study described here we investigated whether the broad-spectrum antiparasitic and antidiarrheal drug nitazoxanide (NTZ) might be active against EAEC in vitro. While E. coli strains were resistant to NTZ in rich Luria-Bertani medium (MIC > 64 μg/ml), the drug was slightly inhibitory in a minimal medium supplemented with glucose (MinA-G medium; MIC, ∼32 μg/ml). NTZ also inhibited biofilm production by strain EAEC 042 in both Dulbecco's modified Eagle's medium and MinA-G medium with a 50% inhibitory concentration of ∼12 μg/ml. Immunofluorescence and immunoblot analyses with antibody against the major fimbrial subunit AafA of aggregative adherence fimbriae vaariant II (AAF/II) established that the numbers of AAF/II filaments on bacteria grown in the presence of NTZ were dramatically reduced. Comparative quantitative reverse transcription-PCR and reporter gene fusions (aafA::phoA) indicated that aafA expression was unaffected by NTZ, while aggR transcript levels and aggR::lacZ expression were increased ∼10- and 2.5-fold, respectively, compared with that for the untreated controls. More generally, NTZ inhibited hemagglutination (HA) of red blood cells by the non-biofilm-producing strain JM221 expressing either AAF/I or type I fimbriae. Our findings suggest that the inhibitory action of NTZ on biofilm formation and HA is likely due to inhibition of fimbrial assembly. Antimicrobial agents that inhibit the assembly or function of fimbrial filaments should be good candidates for the prevention of infection.

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