Tac Promoter

2002 ◽  
Author(s):  
Pieter L. DeHaseth ◽  
David R. Setzer
Keyword(s):  
Tac Promoter

Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors

Gene ◽  
1983 ◽  
Vol 26 (2-3) ◽  
pp. 273-282 ◽  
Author(s):  
Miroslawa M. Bagdasarian ◽  
Egon Amann ◽  
Rudolf Lurz ◽  
Beate Rückert ◽  
Michael Bagdasarian
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Host Range ◽  
Expression Vectors ◽  
Broad Host Range ◽  
Tac Promoter

scholarly journals Characterization of Shiga-like toxin I B subunit purified from overproducing clones of the SLT-I B cistron

1990 ◽  
Vol 272 (3) ◽  
pp. 805-811 ◽  
Author(s):  
K Ramotar ◽  
B Boyd ◽  
G Tyrrell ◽  
J Gariepy ◽  
C Lingwood ◽  
...  
Keyword(s):  
Culture Medium ◽  
Polymyxin B ◽  
Exchange Chromatography ◽  
Structural Studies ◽  
Wild Type ◽  
Binding Studies ◽  
B Subunit ◽  
Tac Promoter ◽  
Glycolipid Receptor

The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background. The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation. Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture. B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing. Cross-linking analysis of purified native B subunit showed that it exists as a pentamer. In gels containing 0.1% SDS the native protein dissociated into monomers. B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin. Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor. We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid.

scholarly journals Properties of human testis-specific lactate dehydrogenase expressed from Escherichia coli

1991 ◽  
Vol 273 (3) ◽  
pp. 587-592 ◽  
Author(s):  
K M LeVan ◽  
E Goldberg
Keyword(s):  
Escherichia Coli ◽  
Lactate Dehydrogenase ◽  
Prokaryotic Expression ◽  
Specific Activity ◽  
Human Testis ◽  
Reading Frame ◽  
E Coli ◽  
Heat Stable ◽  
Prokaryotic Expression Vector ◽  
Tac Promoter

The cDNA encoding the C4 isoenzyme of lactate dehydrogenase (LDH-C4) was engineered for expression in Escherichia coli. The Ldh-c open reading frame was constructed as a cassette for production of the native protein. The modified Ldh-c cDNA was subcloned into the prokaryotic expression vector pKK223-3. Transformed E. coli cells were grown to mid-exponential phase, and induced with isopropyl beta-D-thiogalactopyranoside for positive regulation of the tac promoter. Induced cells expressed the 35 kDa subunit, which spontaneously formed the enzymically active 140 kDa tetramer. Human LDH-C4 was purified over 200-fold from litre cultures of cells by AMP and oxamate affinity chromatography to a specific activity of 106 units/mg. The enzyme was inhibited by pyruvate concentrations above 0.3 mM, had a Km for pyruvate of 0.03 mM, a turnover number (nmol of NADH oxidized/mol of LDH-C4 per min at 25 degrees C) of 14,000 and was heat-stable.

Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli

Gene ◽  
1988 ◽  
Vol 69 (2) ◽  
pp. 301-315 ◽  
Author(s):  
Egon Amann ◽  
Birgit Ochs ◽  
Karl-Josef Abel
Keyword(s):  
Escherichia Coli ◽  
Tac Promoter

scholarly journals Analysis of sequence elements important for expression and regulation of the adenylate cyclase gene (cya) of Salmonella typhimurium.

Genetics ◽  
1990 ◽  
Vol 125 (4) ◽  
pp. 709-717
Author(s):  
L K Thorner ◽  
J P Fandl ◽  
S W Artz
Keyword(s):  
Salmonella Typhimurium ◽  
Structural Gene ◽  
Regulatory Region ◽  
Deletion Mutants ◽  
Stable Secondary Structure ◽  
Translation Initiation Region ◽  
Translation Start ◽  
Sequence Elements ◽  
Tac Promoter ◽  
Delta G

Abstract We determined the nucleotide sequence of the regulatory region of the cya gene of Salmonella typhimurium. A set of nested BAL-31 deletions originating upstream of the promoter/regulatory region and extending into the cya structural gene was constructed in M13mp::cya phages and was tested for complementation of a chromosomal cya deletion mutation. BAL-31 deletion mutants unable to complement cya localized the major cya promoter. The synthetic tac promoter was inserted upstream of the BAL-31 deletions so that expression of cya was dependent on transcription from tac. Those tac derivative phages unable to complement cya localized the translation initiation region. The cya DNA sequence revealed at least three potential promoters capable of transcribing cya, with a CRP binding site straddling the-10 hexamer of the promoter proximal to the structural gene. The leader RNA sequence initiated at the latter promoter is approximately 140 bases long and includes a region that may form a stable secondary structure (delta G = -23.8 kcal). There exist two possible in-frame translation start points, one of which is TTG and the other of which is ATG. The sequence of the S. typhimurium regulatory region was compared with that reported for Escherichia coli.

Expression of Functional Coagulation Factor XIII in Escherichia coli

1990 ◽  
Vol 63 (02) ◽  
pp. 235-240 ◽  
Author(s):  
P G Board ◽  
K Pierce ◽  
M Coggan
Keyword(s):  
Escherichia Coli ◽  
Factor Xiii ◽  
Blood Clotting ◽  
Coagulation Factor ◽  
E Coli ◽  
Thrombin Cleavage ◽  
Coagulation Factor Xiii ◽  
Clotting Cascade ◽  
Tac Promoter ◽  
A Subunit

SummaryCoagulation factor XIII is a zymogen that can be activated by thrombin cleavage to a transglutaminase that catalyses the formation of covalent crosslinks between fibrin chains in the final stages of the blood clotting cascade. Although circulating factor-XIII is composed of A and B subunits the catalytic activity is a property of the A subunits. In this study we have constructed a plasmid (pKKF13A) that contains a cDNA encoding the A subunit positioned downstream of a tac promoter. Escherichia coli containing this plasmid produce A subunit protein when grown in the presence of IPTG. The cloned A subunit has been partially purified and characterized. Comparison with A subunits purified from plasma showed that the cloned A subunits were of the same size, assembled as dimers, and had the same native electrophoretic mobility. The cloned A subunits expressed transglutaminase activity with putrescine, dansylcadaverine and casein as substrates, and were able to crosslink fibrin in clots formed from A subunit deficient plasma. These studies have demonstrated that functional recombinant factor XIII A subunit can be produced in E. coli and suggest that recombinant factor XIII can potentially provide a safe and inexhaustible supply for therapeutic use.

A novel I-TAC promoter polymorphic variant is functional in the presence of replicating HCV in vitro

2005 ◽  
Vol 32 (2) ◽  
pp. 137-143 ◽  
Author(s):  
K.J. Helbig ◽  
J. George ◽  
M.R. Beard
Keyword(s):  
Polymorphic Variant ◽  
Tac Promoter
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