Expression of a human proenkephalin a cDNA in Escherichia coli

1990 ◽  
Vol 10 (5) ◽  
pp. 461-467
Author(s):  
Hans-Jürg Monstein
Keyword(s):  
Escherichia Coli ◽  
Base Pair ◽  
Recombinant Plasmid ◽  
Terminal Part ◽  
E Coli ◽  
Rapid Purification ◽  
Proenkephalin A

A 1000 base pair cDNA coding for the entire human proenkephalin A(proA) polypeptide was subcloned into the multifunctional pMPV 2911/M E. coli vector. The recombinant plasmid was found to express an approximately 30 kDa prohormone, which was recognized by a Met-Arg6-Phe2 antibody, directed against the C-terminal part of the enkephalin A prohormone. The expression of human proenkephalin A cDNA should thus permit the rapid purification of unfused recombinant enkephalin A prohormone, which itself may provide a model substrat to identify endoproteolytic processing activities.

scholarly journals Novel Plasmid-Mediated 16S rRNA m1A1408 Methyltransferase, NpmA, Found in a Clinically Isolated Escherichia coli Strain Resistant to Structurally Diverse Aminoglycosides

2007 ◽  
Vol 51 (12) ◽  
pp. 4401-4409 ◽  
Author(s):  
Jun-ichi Wachino ◽  
Keigo Shibayama ◽  
Hiroshi Kurokawa ◽  
Kouji Kimura ◽  
Kunikazu Yamane ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Recombinant Plasmid ◽  
Coli Strain ◽  
Methyltransferase Activity ◽  
Ribosomal Subunits ◽  
E Coli ◽  
Rrna Molecule ◽  
A Site

ABSTRACT We have isolated a multiple-aminoglycoside-resistant Escherichia coli strain, strain ARS3, and have been the first to identify a novel plasmid-mediated 16S rRNA methyltransferase, NpmA. This new enzyme shared a relatively low level of identity (30%) to the chromosomally encoded 16S rRNA methyltransferase (KamA) of Streptomyces tenjimariensis, an actinomycete aminoglycoside producer. The introduction of a recombinant plasmid carrying npmA could confer on E. coli consistent resistance to both 4,6-disubstituted 2-deoxystreptamines, such as amikacin and gentamicin, and 4,5-disubstituted 2-deoxystreptamines, including neomycin and ribostamycin. The histidine-tagged NpmA elucidated methyltransferase activity against 30S ribosomal subunits but not against 50S subunits and the naked 16S rRNA molecule in vitro. We further confirmed that NpmA is an adenine N-1 methyltransferase specific for the A1408 position at the A site of 16S rRNA. Drug footprinting data indicated that binding of aminoglycosides to the target site was apparently interrupted by methylation at the A1408 position. These observations demonstrate that NpmA is a novel plasmid-mediated 16S rRNA methyltransferase that provides a panaminoglycoside-resistant nature through interference with the binding of aminoglycosides toward the A site of 16S rRNA through N-1 methylation at position A1408.

scholarly journals Na+/H+ antiport activity conferred by Bacillus subtilis tetA(L), a 5' truncation product of tetA(L), and related plasmid genes upon Escherichia coli.

1996 ◽  
Vol 40 (4) ◽  
pp. 852-857 ◽  
Author(s):  
J Cheng ◽  
K Baldwin ◽  
A A Guffanti ◽  
T A Krulwich
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Recombinant Plasmid ◽  
Tetracycline Resistance ◽  
Coding Sequence ◽  
E Coli ◽  
22Na Uptake ◽  
Plasmid Genes ◽  
Related Plasmid ◽  
Is10 Element

An Escherichia coli transformant expressing the Bacillus subtilis tetA(L) gene from a weak promoter was challenged by growth on medium with low, increasing tetracycline concentrations. Changes in the substrate preference ratios of the TetA(L)-mediated resistances and antiports were examined in view of recent findings suggesting that TetA(L) catalyzes efflux of Na+ in exchange for protons in addition to having the ability to catalyze metal-tetracycline/H+ antiport. After growth of the transformant on 1 microgram or more of tetracycline per ml for 12 to 15 h, the tetA(L) gene in the plasmid was found to be disrupted by an IS10 element 50 bp from the 5' end of the coding sequence. This disrupted recombinant plasmid, pKB1, conferred greater tetracycline resistance and higher levels of membrane metal-tetracycline/proton antiport than the original plasmid, pJTA1, but conferred lower NA+ resistance and Na+/H+ antiport levels than the original plasmid. The results indicate that the 5' end of the gene is necessary for optimal Na+/H+ antiport but that some such activity as well as robust tetracycline/H+ antiport persists in its absence. Two plasmid genes, tet(K) and qacA, were compared with tetA(L) vis-à-vis their abilities to enhance the Na+/H+ antiporter activity of everted vesicles from E. coli transformants. tet(K), which is more closely related to tetA(L), catalyzed 22Na+ uptake by energized vesicles, whereas the less closely related qacA gene did not.

scholarly journals Expression comparison between two genes encoding CSF3 recombinant proteins having different codon composition at N-terminal in Escherichia coli

2021 ◽  
Vol 948 (1) ◽  
pp. 012081
Author(s):  
K S Dewi ◽  
F D Wahyuni ◽  
S Salsabila ◽  
Aminah ◽  
N D Yanthi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Recombinant Proteins ◽  
Recombinant Plasmid ◽  
Synonymous Codon ◽  
Expression System ◽  
Negative Control ◽  
Translation Initiation Region ◽  
E Coli ◽  
Genes Encoding

Abstract Colony-stimulating factor 3 (CSF3) is a glycoprotein with many therapeutic applications. In the Escherichia coli expression system, mRNA folding and stability near the translation initiation region (TIR) are known to influence protein expression significantly. We have successfully constructed the recombinant plasmid carrying genes encoding CSF3.1 and CSF3.2, which have different synonymous codon usage at N-terminal. In this study, we compared both expressions of CSF3.1 and CSF3.2 recombinant proteins in E. coli host. Recombinant plasmid pJ414-CSF3.1 and pJ414-CSF3.2 were transformed individually into E. coli NiCo21(DE3) competent cells by a heat-shock method, then spread on solid Lysogeny Broth (LB) medium containing ampicillin. Eight transformant colonies were selected and then expressed in 2xYT medium with the addition of IPTG inducer. Expression analysis was carried out using 15% SDS-PAGE gel. No significantly different band was observed in CSF3.1 protein expression compared to the negative control. In contrast, CSF3.2 protein can be expressed with a good amount at its expected size of 18 kDa. This result was strengthened by bioinformatics analysis which demonstrated the more open TIR of CSF3.2 than that of CSF3.1 Our study highlighted that AU-rich mRNA at the N-terminal is essential for efficient recognition of the ribosome binding site.

Overproduction of the Bacillus subtilis glutamyl-tRNA synthetase in its host and its toxicity to Escherichia coli

1998 ◽  
Vol 44 (4) ◽  
pp. 378-381 ◽  
Author(s):  
Martin Pelchat ◽  
Lucille Lacoste ◽  
Fu Yang ◽  
Jacques Lapointe
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Recombinant Plasmid ◽  
Specific Activity ◽  
Shuttle Vector ◽  
Fold Increase ◽  
Trna Synthetase ◽  
E Coli ◽  
Downstream Region

The Bacillus subtilis glutamyl-tRNA synthetase (GluRS), encoded by the gltX gene, aminoacylates its homologous tRNAGlu and tRNAGln with glutamate. This gene was cloned with its sigmaA promoter and a downstream region including a rho-independent terminator in the shuttle vector pRB394 for Escherichia coli and B. subtilis. Transformation of B. subtilis with this recombinant plasmid (pMP411) led to a 30-fold increase of glutamyl-tRNA synthetase specific activity in crude extracts. Transformation of E. coli with this plasmid gave no recombinants, but transformation with plasmids bearing an altered gltX was successful. These results indicate that the presence of B. subtilis glutamyl-tRNA synthetase is lethal for E. coli, probably because this enzyme glutamylates tRNA1Gln in vivo as it does in vitro.Key words: glutamyl-tRNA synthetase overproduction, Bacillus subtilis, toxicity, Escherichia coli.

scholarly journals A single base pair dominates over the novel identity of an Escherichia coli tyrosine tRNA in Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (5) ◽  
pp. 2744-2751 ◽  
Author(s):  
V Trézéguet ◽  
H Edwards ◽  
P Schimmel
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Major Determinant ◽  
The Novel ◽  
Single Base ◽  
E Coli ◽  
Single Base Pair

The Escherichia coli su+3 tyrosine tRNA was shown recently to be a leucine-specific tRNA in Saccharomyces cerevisiae. This finding raises the possibility that some determinants for tRNA identity in E. coli may be different in S. cerevisiae. To investigate whether the fungal system is sensitive to the major determinant for alanine acceptance in E. coli, a single G3 . U70 base pair was introduced into the acceptor helix of the su+3 tyrosine tRNA. This substitution converts the identity of the E. coli suppressor in S. cerevisiae from leucine to alanine. Thus, as in E. coli, G3 . U70 is a strong determinant for alanine acceptance that can dominate over other features in a tRNA that might be recognized by alternative charging enzymes.

scholarly journals Evaluation of Pathogenic Potentials of Microbial Contaminants from Naira Notes in Nigeria

2021 ◽  
pp. 180-188
Author(s):  
Ifeoma Bessie Enweani ◽  
Chinyere Nkemjika Anyanwu ◽  
Emmanuel Ifeanyi Obeagu
Keyword(s):  
Escherichia Coli ◽  
Aspergillus Niger ◽  
Proteus Mirabilis ◽  
Base Pair ◽  
Alveolar Epithelium ◽  
Local Alignment ◽  
Histopathological Study ◽  
E Coli ◽  
Watery Stool ◽  
Search Tool

The study was done to determine burden of fiat currencies. A total of Six hundred and twenty four pieces of different denominations of naira notes obtained from banks in Enugu metropolis and samples of nose swabs aseptically collected from fifty two note counters from those banks were examined for similar bacterial and fungal contaminants. All sequences were identified using the Basic Local Alignment Search Tool (BLAST) on National Center for Biotechnology Information (NCBI) website. While the fungi amplicons yielded DNA bands of approximately 650 base pair, that of the bacterial isolates were approximately 850 base pair. Proteus mirabilis (NR11449.1) and Escherichia coli (LN831043.1) were identical and selected from the bacterial category while Aspergillus fumigatus (MK910068), Aspergillus flavus (JQ860302) and Aspergillus niger (MK461093) were identical and selected from the fungal category. Rats inoculated orally with Escherichia coli and Proteus mirabilis presented with watery stool and reduction of weight by 16+0.4g after two weeks of commencement of inoculation. They showed reduction in activity and reduced locomotion when compared with the control. There were no physically observable changes in the other test groups. In the hematological investigation, the mean PCV in % were 39±1.0 for the E. coli, 40±0.4 for P. mirabilis, 35±0.2 for A. niger, 40±0.7 for A. fumigatus and 37±0.1 for A .flavus. These varied significantly at p<0.05 with the control which has mean PCV of 45±0.3. The differential leucocyte count showed a marked increase in the % neutrophil (E. coli 73±0.1, P. mirabilis 70±0.1, A. niger 78±1.1, A. fumigatus 59±0.3 and A. flavus 62±1.0) when compared with the control rats with percentage neutrophil of 20±0.2. There was also an increase in the white blood cell count of the test groups when compared with the control. Histopathological study of the lungs of the rats inoculated nasally with Aspergillus niger showed necrosis of the alveolar epithelium. This study has shown that naira notes could be a reservoir of microorganisms of medical importance which in turn could become vectors for the transmission of diseases in the society.

scholarly journals Cloning and expression of a deoxyribonucleic acid fragment that encodes for the adhesive antigen K99

1980 ◽  
Vol 29 (3) ◽  
pp. 1125-1133
Author(s):  
J D van Embden ◽  
F K de Graaf ◽  
L M Schouls ◽  
J S Teppema
Keyword(s):  
Escherichia Coli ◽  
Recombinant Plasmid ◽  
Deoxyribonucleic Acid ◽  
Cloning And Expression ◽  
Morphological Properties ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Naturally Occurring ◽  
Acid Fragment ◽  
K 12

Deoxyribonucleic acid fragments of the naturally occurring conjugative K99 plasmid were cloned into vectors pBR322 and pBR325. The smallest deoxyribonucleic acid segment obtained that still expressed K99 was 4.5 megadaltons in size. With regard to the serological, adhesive, and morphological properties, no differences in the nature of the K99 antigen was observed between Escherichia coli strains carrying recombinant plasmids and those carrying pRI9901. Furthermore, the regulation of K99 expression from the recombinant plasmid deoxyribonucleic acid was similar to that from pRI9901. the low level of K99 expression in E. coli K-12 compared with natural K99-producing isolates seems to be host specific.

scholarly journals Some Features of Base Pair Mismatch and Heterology Repair in Escherichia coli

Genetics ◽  
1987 ◽  
Vol 117 (3) ◽  
pp. 381-390
Author(s):  
Susan Raposa ◽  
Maurice S Fox
Keyword(s):  
Escherichia Coli ◽  
Mismatch Repair ◽  
Base Pair ◽  
Repair System ◽  
Loop Structure ◽  
E Coli ◽  
Base Pair Mismatch ◽  
Adenine Methylation ◽  
Mismatch Repair System ◽  
Lambda Dna

ABSTRACT We have used artificially constructed heteroallelic heteroduplex molecules of bacteriophage lambda DNA to transfect Escherichia coli, and E. coli mutants deficient in various functions involved in the adenine methylation-directed mismatch repair system, MutL, MutS, MutH, and UvrD (MutU). Analysis of the allele content of single infective centers shows that this repair system often acts on several mismatches, separated by as many as 2000 bp, on one of the strands of a heteroduplex molecule. When the methyl-directed mismatch repair system is disabled by mutH or uvrD mutations, localized mismatch repair becomes prominent. This prominent localized repair that can result in separation of very closely linked markers requires the functions MutL and MutS, is independent of adenine methylation, and appears to reflect another mechanism of mismatch repair. Heterology-containing heteroduplex molecules with a deletion in one strand often escape processing. However, when the heterology includes the stem and loop structure of a transposon, Tn10, the transposon is lost.

A single base pair dominates over the novel identity of an Escherichia coli tyrosine tRNA in Saccharomyces cerevisiae

1991 ◽  
Vol 11 (5) ◽  
pp. 2744-2751
Author(s):  
V Trézéguet ◽  
H Edwards ◽  
P Schimmel
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Base Pair ◽  
Major Determinant ◽  
The Novel ◽  
Single Base ◽  
E Coli ◽  
Single Base Pair

The Escherichia coli su+3 tyrosine tRNA was shown recently to be a leucine-specific tRNA in Saccharomyces cerevisiae. This finding raises the possibility that some determinants for tRNA identity in E. coli may be different in S. cerevisiae. To investigate whether the fungal system is sensitive to the major determinant for alanine acceptance in E. coli, a single G3 . U70 base pair was introduced into the acceptor helix of the su+3 tyrosine tRNA. This substitution converts the identity of the E. coli suppressor in S. cerevisiae from leucine to alanine. Thus, as in E. coli, G3 . U70 is a strong determinant for alanine acceptance that can dominate over other features in a tRNA that might be recognized by alternative charging enzymes.

scholarly journals Purification and characterization of glutathione reductase encoded by a cloned and over-expressed gene in Escherichia coli

1987 ◽  
Vol 245 (3) ◽  
pp. 875-880 ◽  
Author(s):  
N S Scrutton ◽  
A Berry ◽  
R N Perham
Keyword(s):  
Escherichia Coli ◽  
Glutathione Reductase ◽  
Reductase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Fold Increase ◽  
Site Directed Mutagenesis ◽  
E Coli ◽  
Glutathione Reductase Activity ◽  
Rapid Purification ◽  
Sepharose 4B

An expression vector, pKGR, for the gor gene from Escherichia coli encoding glutathione reductase was constructed by subcloning of an AvaII fragment of the Clarke & Carbon bank plasmid pGR [Greer & Perham (1986) Biochemistry 25, 2736-2742] into the plasmid pKK223-3. The expression of glutathione reductase from the plasmid pKGR was found to have been successfully placed under the control of the tac promoter. Transformation of E. coli cells with this plasmid resulted in 100-200-fold increase in glutathione reductase activity in cell-free extracts. A rapid purification procedure for the enzyme, based on affinity chromatography on Procion Red HE-7B-CL-Sepharose 4B, was developed. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-gel electrophoresis, and all its properties were consistent with the DNA sequence of the gene [Greer & Perham (1986) Biochemistry 25, 2736-2742] and with those previously reported for E. coli glutathione reductase [Mata, Pinto & Lopez-Barea (1984) Z. Naturforsch. C. Biosci. 39, 908-915]. These experiments have enabled an investigation of the protein chemical and mechanistic properties of the enzyme by site-directed mutagenesis.

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