scholarly journals Troubleshooting coupled in vitro transcription–translation system derived from Escherichia coli cells: synthesis of high-yield fully active proteins

2006 ◽  
Vol 34 (19) ◽  
pp. e135-e135 ◽  
Author(s):  
Madina B. Iskakova ◽  
Witold Szaflarski ◽  
Marc Dreyfus ◽  
Jaanus Remme ◽  
Knud H. Nierhaus
Keyword(s):  
Escherichia Coli ◽  
In Vitro Transcription ◽  
High Yield ◽  
Translation System ◽  
Escherichia Coli Cells

scholarly journals The Chromosomal Arsenic Resistance Genes of Thiobacillus ferrooxidans Have an Unusual Arrangement and Confer Increased Arsenic and Antimony Resistance to Escherichia coli

2000 ◽  
Vol 66 (5) ◽  
pp. 1826-1833 ◽  
Author(s):  
Bronwyn G. Butcher ◽  
Shelly M. Deane ◽  
Douglas E. Rawlings
Keyword(s):  
Escherichia Coli ◽  
Resistance Genes ◽  
Thiobacillus Ferrooxidans ◽  
In Vitro Transcription ◽  
Translation System ◽  
Gram Positive ◽  
Arsenic Resistance ◽  
E Coli ◽  
Gram Staining

ABSTRACT The chromosomal arsenic resistance genes of the acidophilic, chemolithoautotrophic, biomining bacterium Thiobacillus ferrooxidans were cloned and sequenced. Homologues of four arsenic resistance genes, arsB, arsC,arsH, and a putative arsR gene, were identified. The T. ferrooxidans arsB (arsenite export) andarsC (arsenate reductase) gene products were functional when they were cloned in an Escherichia coli ars deletion mutant and conferred increased resistance to arsenite, arsenate, and antimony. Therefore, despite the fact that the ars genes originated from an obligately acidophilic bacterium, they were functional in E. coli. Although T. ferrooxidansis gram negative, its ArsC was more closely related to the ArsC molecules of gram-positive bacteria. Furthermore, a functionaltrxA (thioredoxin) gene was required for ArsC-mediated arsenate resistance in E. coli; this finding confirmed the gram-positive ArsC-like status of this resistance and indicated that the division of ArsC molecules based on Gram staining results is artificial. Although arsH was expressed in an E. coli-derived in vitro transcription-translation system, ArsH was not required for and did not enhance arsenic resistance in E. coli. The T. ferrooxidans ars genes were arranged in an unusual manner, and the putative arsR andarsC genes and the arsBH genes were translated in opposite directions. This divergent orientation was conserved in the four T. ferrooxidans strains investigated.

Cloning the gene for the heat shock response positiwe regulator (sigma 32 homolog) from Pseudomonas aeruginosa

1995 ◽  
Vol 41 (1) ◽  
pp. 75-87 ◽  
Author(s):  
Zerlina M. Naczynski ◽  
Andrew M. Kropinski ◽  
Chris Mueller
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Heat Shock ◽  
Sigma Factor ◽  
Oligonucleotide Probe ◽  
In Vitro Transcription ◽  
Translation System ◽  
Amino Acid Homology ◽  
E Coli ◽  
Transcriptional Start Sites

A 31 base pair synthetic oligonucleotide based on the genes for the Escherichia coli heat shock sigma factor (rpoH) and the Pseudomonas aeruginosa housekeeping sigma factor (rpoD) was employed in conjunction with the Tanaka et al. (K. Tanaka, T. Shiina, and H. Takahashi, 1988. Science (Washington, D.C.), 242: 1040–1042) RpoD box probe to identify the location of the rpoH gene in P. aeruginosa genomic digests. This gene was cloned into plasmid pGEM3Z(f+), sequenced, and found to share 67% nucleotide identity and 77% amino acid homology with the rpoH gene and its product (σ32) of E. coli. The plasmid containing the rpoH gene complemented the function of σ32 in an E. coli rpoH deletion mutant. Furthermore, this plasmid directed the synthesis of a 32-kDa protein in an E. coli S-30 in vitro transcription–translation system. Primer extension studies were used to identify the transcriptional start sites under control and heat-stressed (45 and 50 °C) conditions. Two promoter sites were identified having sequence homology to the E. coli σ70 and σ24 consensus sequences.Key words: heat shock, Pseudomonas aeruginosa, sigma factor, transcription, oligonucleotide probe.

scholarly journals Identification of an AU-rich Translational Enhancer within the Escherichia coli fepB Leader RNA

2007 ◽  
Vol 189 (11) ◽  
pp. 4028-4037 ◽  
Author(s):  
India G. Hook-Barnard ◽  
Timothy J. Brickman ◽  
Mark A. McIntosh
Keyword(s):  
Escherichia Coli ◽  
Posttranscriptional Regulation ◽  
Selective Advantage ◽  
In Vitro Transcription ◽  
Start Codon ◽  
Translational Efficiency ◽  
Rnase Protection ◽  
Ribosome Binding ◽  
Translational Enhancer

ABSTRACT The fepB gene encodes a periplasmic binding protein that is essential for the uptake of ferric enterobactin by Escherichia coli. Its transcription is regulated in response to iron levels by the Fur repressor. The fepB transcript includes a 217-nucleotide leader sequence with several features suggestive of posttranscriptional regulation. To investigate the fepB leader for its contribution to fepB expression, defined deletions and substitution mutations in the leader were characterized using fepB-phoA translational fusions. The fepB leader was found to be necessary for maximal fepB expression, primarily due to the influence of an AU-rich translational enhancer (TE) located 5′ to the Shine-Dalgarno sequence. Deletions or substitutions within the TE sequence decreased fepB-phoA expression fivefold. RNase protection and in vitro transcription-translation assays demonstrated that the TE augmented translational efficiency, as well as RNA levels. Moreover, primer extension inhibition assays showed that the TE increases ribosome binding. In contrast to the enhancing effect of the TE, the natural fepB GUG start codon decreased ribosome binding and reduced fepB expression 2.5-fold compared with the results obtained with leaders bearing an AUG initiation codon. Thus, the TE-GUG organization in fepB results in an intermediate level of expression compared to the level with AUG, with or without the TE. Furthermore, we found that the TE-GUG sequence is conserved among the eight gram-negative strains examined that have fepB genes, suggesting that this organization may provide a selective advantage.

scholarly journals Aglycosylated antibodies and antibody fragments produced in a scalable in vitro transcription-translation system

mAbs ◽  
2012 ◽  
Vol 4 (2) ◽  
pp. 217-225 ◽  
Author(s):  
Gang Yin ◽  
Eudean D. Garces ◽  
Junhao Yang ◽  
Juan Zhang ◽  
Cuong Tran ◽  
...  
Keyword(s):  
In Vitro Transcription ◽  
Antibody Fragments ◽  
Translation System

scholarly journals Identification of a talin binding site in the cytoskeletal protein vinculin.

1989 ◽  
Vol 109 (6) ◽  
pp. 2917-2927 ◽  
Author(s):  
P Jones ◽  
P Jackson ◽  
G J Price ◽  
B Patel ◽  
V Ohanion ◽  
...  
Keyword(s):  
Amino Acids ◽  
In Vitro Transcription ◽  
Binding Activity ◽  
Cytoskeletal Protein ◽  
Translation System ◽  
Cos Cells ◽  
Cell Matrix ◽  
Do So

Binding of the cytoskeletal protein vinculin to talin is one of a number of interactions involved in linking F-actin to cell-matrix junctions. To identify the talin binding domain in vinculin, we expressed the NH2-terminal region of the molecule encoded by two closely similar, but distinct vinculin cDNAs, using an in vitro transcription translation system. The 5' Eco RI-Bam HI fragment of a partial 2.89-kb vinculin cDNA encodes a 45-kD polypeptide containing the first 398 amino acids of the molecule. The equivalent restriction enzyme fragment of a second vinculin cDNA (cVin5) lacks nucleotides 746-867, and encodes a 41-kD polypeptide missing amino acids 167-207. The radiolabeled 45-kD vinculin polypeptide bound to microtiter wells coated with talin, but not BSA, and binding was inhibited by unlabeled vinculin. In contrast, the 41-kD vinculin polypeptide was devoid of talin binding activity. The role of residues 167-207 in talin binding was further analyzed by making a series of deletions spanning this region, each deletion of seven amino acids contiguous with the next. Loss of residues 167-173, 174-180, 181-187, 188-194, or 195-201 resulted in a marked reduction in talin binding activity, although loss of residues 202-208 had much less effect. When the 45-kD vinculin polypeptide was expressed in Cos cells, it localized to cell matrix junctions, whereas the 41-kD polypeptide, lacking residues 167-207, was unable to do so. Interestingly, some deletion mutants with reduced ability to bind talin in vitro, were still able to localize to cell matrix junctions.

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