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.

scholarly journals Identification and Function of the pdxY Gene, Which Encodes a Novel Pyridoxal Kinase Involved in the Salvage Pathway of Pyridoxal 5′-Phosphate Biosynthesis in Escherichia coli K-12

1998 ◽  
Vol 180 (7) ◽  
pp. 1814-1821 ◽  
Author(s):  
Yong Yang ◽  
Ho-Ching Tiffany Tsui ◽  
Tsz-Kwong Man ◽  
Malcolm E. Winkler
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Specific Activity ◽  
Salvage Pathway ◽  
Pyridoxal Kinase ◽  
Triple Mutant ◽  
Reading Frame ◽  
E Coli ◽  
Specific Activities ◽  
K 12

ABSTRACT pdxK encodes a pyridoxine (PN)/pyridoxal (PL)/pyridoxamine (PM) kinase thought to function in the salvage pathway of pyridoxal 5′-phosphate (PLP) coenzyme biosynthesis. The observation that pdxK null mutants still contain PL kinase activity led to the hypothesis that Escherichia coli K-12 contains at least one other B6-vitamer kinase. Here we support this hypothesis by identifying the pdxY gene (formally, open reading frame f287b) at 36.92 min, which encodes a novel PL kinase. PdxY was first identified by its homology to PdxK in searches of the complete E. coli genome. Minimal clones of pdxY + overexpressed PL kinase specific activity about 10-fold. We inserted an omega cassette intopdxY and crossed the resultingpdxY::ΩKanr mutation into the bacterial chromosome of a pdxB mutant, in which de novo PLP biosynthesis is blocked. We then determined the growth characteristics and PL and PN kinase specific activities in extracts ofpdxK and pdxY single and double mutants. Significantly, the requirement of the pdxB pdxK pdxY triple mutant for PLP was not satisfied by PL and PN, and the triple mutant had negligible PL and PN kinase specific activities. Our combined results suggest that the PL kinase PdxY and the PN/PL/PM kinase PdxK are the only physiologically important B6vitamer kinases in E. coli and that their function is confined to the PLP salvage pathway. Last, we show thatpdxY is located downstream from pdxH (encoding PNP/PMP oxidase) and essential tyrS (encoding aminoacyl-tRNATyr synthetase) in a multifunctional operon.pdxY is completely cotranscribed with tyrS, but about 92% of tyrS transcripts terminate at a putative Rho-factor-dependent attenuator located in thetyrS-pdxY intercistronic region.

Overproduction in Escherichia coli of the dehydroquinate synthase domain of the Aspergillus nidulans pentafunctional AROM protein

1992 ◽  
Vol 284 (3) ◽  
pp. 861-867 ◽  
Author(s):  
J P T W van den Hombergh ◽  
J D Moore ◽  
I G Charles ◽  
A R Hawkins
Keyword(s):  
Escherichia Coli ◽  
Aspergillus Nidulans ◽  
Dna Sequences ◽  
Specific Activity ◽  
Stop Codon ◽  
Cell Protein ◽  
Shikimate Dehydrogenase ◽  
E Coli ◽  
Prokaryotic Expression Vector ◽  
Dehydroquinate Synthase

The pentafunctional AROM protein of Aspergillus nidulans is encoded by the complex aromA locus and catalyses steps 2-6 in the synthesis of chorismate, the common precursor for the aromatic amino acids and p-aminobenzoic acid. DNA sequences encoding the 3-dehydroquinate synthase (DHQ synthase) and 3-dehydroquinase domains of the AROM protein have been amplified with the inclusion of a translational stop codon at the C-terminus by PCR technology. These amplified fragments of DNA have been subcloned into the prokaryotic expression vector pKK233-2 and expressed in Escherichia coli. As a result, the DHQ synthase domain is overproduced in E. coli, forming 30% of total cell protein, and can be purified to greater than 80% homogeneity by a simple two-step protocol. The 3-dehydroquinase domain is produced at a specific activity 8-fold greater than the corresponding activity encoded by the aromA gene in A. nidulans. The qutB gene of A. nidulans encoding quinate dehydrogenase has similarly been subjected to PCR amplification and expression in E. coli. The quinate dehydrogenase is not overproduced, but is active in E. coli as a shikimate dehydrogenase, as the presence of the qutB gene allows the growth of an E. coli mutant strain lacking shikimate dehydrogenase on minimal medium lacking aromatic-amino-acid supplementation.

scholarly journals Construction of Deoxyriboaldolase-Overexpressing Escherichia coli and Its Application to 2-Deoxyribose 5-Phosphate Synthesis from Glucose and Acetaldehyde for 2′-Deoxyribonucleoside Production

2003 ◽  
Vol 69 (7) ◽  
pp. 3791-3797 ◽  
Author(s):  
Nobuyuki Horinouchi ◽  
Jun Ogawa ◽  
Takafumi Sakai ◽  
Takako Kawano ◽  
Seiichiro Matsumoto ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Specific Activity ◽  
Cell Extract ◽  
Predicted Amino Acid Sequence ◽  
Enzymatic Reactions ◽  
Chromosomal Dna ◽  
Gene Encoding ◽  
Reading Frame ◽  
E Coli

ABSTRACT The gene encoding a deoxyriboaldolase (DERA) was cloned from the chromosomal DNA of Klebsiella pneumoniae B-4-4. This gene contains an open reading frame consisting of 780 nucleotides encoding 259 amino acid residues. The predicted amino acid sequence exhibited 94.6% homology with the sequence of DERA from Escherichia coli. The DERA of K. pneumoniae was expressed in recombinant E. coli cells, and the specific activity of the enzyme in the cell extract was as high as 2.5 U/mg, which was threefold higher than the specific activity in the K. pneumoniae cell extract. One of the E. coli transformants, 10B5/pTS8, which had a defect in alkaline phosphatase activity, was a good catalyst for 2-deoxyribose 5-phosphate (DR5P) synthesis from glyceraldehyde 3-phosphate and acetaldehyde. The E. coli cells produced DR5P from glucose and acetaldehyde in the presence of ATP. Under the optimal conditions, 100 mM DR5P was produced from 900 mM glucose, 200 mM acetaldehyde, and 100 mM ATP by the E. coli cells. The DR5P produced was further transformed to 2′-deoxyribonucleoside through coupling the enzymatic reactions of phosphopentomutase and nucleoside phosphorylase. These results indicated that production of 2′-deoxyribonucleoside from glucose, acetaldehyde, and a nucleobase is possible with the addition of a suitable energy source, such as ATP.

scholarly journals Prokaryotic Expression, Purification and Identification of Human PDE9A Protein

2018 ◽  
Vol 10 (1) ◽  
pp. 22
Author(s):  
Huan Rui ◽  
Liqun Wang
Keyword(s):  
Prokaryotic Expression ◽  
Expression Vector ◽  
Cyclic Guanosine Monophosphate ◽  
Culture Conditions ◽  
Guanosine Monophosphate ◽  
Reading Frame ◽  
E Coli ◽  
Prokaryotic Expression Vector ◽  
Solid Foundation ◽  
Polymerase Chain

Cerebral aggregation of beta amyloid plaques (Aβ) and neurofibrillary tangles is responsible for the onset of Alzheimer’s disease (AD), and PDE9A inhibition rescues Aβ-induced deficits in synaptic plasticity and cognition. This study was aimed to express active PDE9A protein for subsequent inhibitor screening. The PDE9A gene was cloned from human cDNA by real-time polymerase chain reaction, and then the gene sequence and its amino acid sequence were analyzed on Lasergene. An inducible expression vector was constructed by enzyme digestion-seamless cloning and transformed into Escherichia coli BL21 (DE3) for PDE9A expression with isopropyl β-D-1-thiogalactopyranoside (IPTG) as an inducer. The recombinant protein was purified by Ni-NTA affinity chromatography and its activity was determined by a phosphodiesterase assay kit. It was found the open reading frame of PED9A was 1035 bp long, the deduced protein was composed of 345 amino acids, and its predicted isoelectric point was about 4.84. The E. coli vector ST6-PDE9A successfully expressed the recombinant PDE9A protein in the supernatant of bacterial lysate. The optimal culture conditions were that the bacterium ST6-PDE9A was grown first in a lysogeny broth at 37 ºC to an OD600 of 0.6-0.8 and then at 16 ºC for 40 h with the addition of 1 M IPTG. Activity test showed PDE9A significantly hydrolyzed the substrate cyclic guanosine monophosphate. In conclusion, we constructed a prokaryotic expression vector and expressed active proteins, laying a solid foundation for screening PDE9 inhibitors.

scholarly journals Application of a Propionyl Coenzyme A Synthetase for Poly(3-Hydroxypropionate-co-3-Hydroxybutyrate) Accumulation in Recombinant Escherichia coli

2000 ◽  
Vol 66 (12) ◽  
pp. 5253-5258 ◽  
Author(s):  
Henry E. Valentin ◽  
Timothy A. Mitsky ◽  
Debbie A. Mahadeo ◽  
Minhtien Tran ◽  
Kenneth J. Gruys
Keyword(s):  
Escherichia Coli ◽  
Coenzyme A ◽  
Ralstonia Eutropha ◽  
Specific Activity ◽  
Biosynthetic Genes ◽  
Acetyl Coa ◽  
Reading Frame ◽  
E Coli ◽  
Coa Transferase ◽  
Serovar Typhimurium

ABSTRACT The genetic operon for propionic acid degradation inSalmonella enterica serovar Typhimurium contains an open reading frame designated prpE which encodes a propionyl coenzyme A (propionyl-CoA) synthetase (A. R. Horswill and J. C. Escalante-Semerena, Microbiology 145:1381–1388, 1999). In this paper we report the cloning of prpE by PCR, its overexpression in Escherichia coli, and the substrate specificity of the enzyme. When propionate was utilized as the substrate for PrpE, a Km of 50 μM and a specific activity of 120 μmol � min−1 � mg−1 were found at the saturating substrate concentration. PrpE also activated acetate, 3-hydroxypropionate (3HP), and butyrate to their corresponding coenzyme A esters but did so much less efficiently than propionate. When prpE was coexpressed with the polyhydroxyalkanoate (PHA) biosynthetic genes from Ralstonia eutropha in recombinant E. coli, a PHA copolymer containing 3HP units accumulated when 3HP was supplied with the growth medium. To compare the utility of acyl-CoA synthetases to that of an acyl-CoA transferase for PHA production, PHA-producing recombinant strains were constructed to coexpress the PHA biosynthetic genes with prpE, with acoE (an acetyl-CoA synthetase gene from R. eutropha [H. Priefert and A. Steinb�chel, J. Bacteriol. 174:6590–6599, 1992]), or with orfZ (an acetyl-CoA:4-hydroxybutyrate-CoA transferase gene from Clostridium propionicum [H. E. Valentin, S. Reiser, and K. J. Gruys, Biotechnol. Bioeng. 67:291–299, 2000]). Of the three enzymes, PrpE and OrfZ enabled similar levels of 3HP incorporation into PHA, whereas AcoE was significantly less effective in this capacity.

Comparison of colony and stool blots for detection of enteropathogens by DNA probes

1991 ◽  
Vol 37 (5) ◽  
pp. 407-410
Author(s):  
Mônica A. M. Vieira ◽  
Beatriz E. C. Guth ◽  
Tânia A. T. Gomes
Keyword(s):  
Escherichia Coli ◽  
Key Words ◽  
Sensitivity And Specificity ◽  
Dna Probes ◽  
Type I ◽  
Enteropathogenic Escherichia Coli ◽  
Key Words Dna ◽  
E Coli ◽  
Heat Stable

DNA probes that identify genes coding for heat-labile type I (LT-I) and heat-stable type 1 (ST-I) enterotoxins, enteropathogenic Escherichia coli adherence factor (EAF), and Shigella-like, invasiveness (INV) are used to evaluate the sensitivity and specificity of stool blots in comparison with the sensitivity and specificity of colony blots in detecting enteropathoghens. The sensitivities of the probes in stool blots are 91.7% for the LT-I probe, 76.9% for the ST-I probes, 78.9% for the EAF probe, and 45.5% for the INV probe. The specificity of all probes is higher than 95%. In general, the stool blot method identifies as many if not more LT-I-, ST-I-, and EAF-producing E. coli infections than the colony blots. Key words: DNA probes, stool blots, enteropathogens, diagnosis.

scholarly journals Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

1998 ◽  
Vol 64 (12) ◽  
pp. 4891-4896 ◽  
Author(s):  
Ji Qiu ◽  
James R. Swartz ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Plasminogen Activator ◽  
Disulfide Bonds ◽  
Specific Activity ◽  
Active Form ◽  
Tissue Type ◽  
Heterologous Proteins ◽  
E Coli ◽  
Disulfide Isomerases

ABSTRACT The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins inE. coli without the need for in vitro refolding.

scholarly journals Erratum

2003 ◽  
Vol 130 (3) ◽  
pp. 573-573
Author(s):  
Z. ZHOU ◽  
J. OGASAWARA ◽  
Y. NISHIKAWA
Keyword(s):  
Escherichia Coli ◽  
E Coli ◽  
Heat Stable ◽  
Epidemiol Infect

Epidemiol. Infect. 128 (2002), 363–371An outbreak of gastroenteritis in Osaka, Japan due toEscherichia coliserogroup O166[ratio ]H15 that had a coding gene for enteroaggregativeE. coliheat-stable enterotoxin 1 (EAST1)Tables 1 and 2 were omitted

scholarly journals Evolution of diarrheagenic Escherichia coli pathotypes in India

2019 ◽  
Vol 11 (04) ◽  
pp. 346-351
Author(s):  
Pankaj Singh ◽  
Sharda C. Metgud ◽  
Subarna Roy ◽  
Shashank Purwar
Keyword(s):  
Escherichia Coli ◽  
Cross Sectional Study ◽  
Clinical Settings ◽  
Accurate Identification ◽  
Cross Sectional ◽  
E Coli ◽  
Medical College ◽  
Diarrheagenic Escherichia Coli ◽  
Proper Management ◽  
Heat Stable

Abstract CONTEXT: Diarrheagenic Escherichia coli (DEC) is the leading cause of infectious diarrhea in developing countries. On the basis of virulence and phenotypic characteristics, the DEC is categorized into multiple pathotypes. Each pathotype has different pathogenesis and geographical distribution. Thus, the proper management of disease relies on rapid and accurate identification of DEC pathotypes. AIMS: The aim of the study was to determine the prevalence of DEC pathotypes in India. MATERIALS AND METHODS: A cross-sectional study was carried out between January 2008 and December 2012 at Jawaharlal Nehru Medical College and KLES Dr. Prabhakar Kore Hospital and Medical Research Center, Belgaum (Karnataka), India. A total of 300 stool samples were collected from diarrhea patients with age >3 months. The DEC was identified by both conventional and molecular methods. RESULTS: Of 300 samples, E. coli were detected in 198 (66%) and 170 (56.6%) samples by culture and polymerase chain reaction, respectively. Among DEC (n = 198) isolates, eae gene (59.5%) was the most prevalent followed by stx (27.7%), east (27.2%), elt (12.6%), est (10.6%), ipaH (5.5%), and eagg (1.5%) genes. On the basis of virulence genes, enteropathogenic E. coli (33.8%) was the most common pathotype followed by Shiga toxin-producing E. coli (STEC, 23.2%), enterotoxigenic E. coli (ETEC, 13.6%), enteroinvasive E. coli (5.5%), enteroaggregative heat-stable enterotoxin 1-harboring E. coli (EAST1EC, 4.5%), STEC/ETEC (3.5%), STEC/enteroaggregative E. coli (STEC/EAEC, 1.0%), and EAEC (0.05%). CONCLUSIONS: The hybrid DEC is potentially more virulent than basic pathotypes. The pathotyping should be included in clinical settings for the proper management of DEC-associated diarrhea.

Purification by Affinity Chromatography of Glutathione Reductase (EC 1.6.4.2) from Escherichia coli and Characterization of such Enzyme

1984 ◽  
Vol 39 (9-10) ◽  
pp. 908-915 ◽  
Author(s):  
Anna M. Mata ◽  
M. Carmen ◽  
Juan López-Barea
Keyword(s):  
Escherichia Coli ◽  
Affinity Chromatography ◽  
Glutathione Reductase ◽  
Specific Activity ◽  
Total Yield ◽  
Coli Strain ◽  
Affinity Column ◽  
Heat Stable ◽  
Activity Loss ◽  
Fast Procedure

Abstract The glutathione reductase from Escherichia coli strain S33 was purified to homogeneity by a simple and fast procedure consisting of two affinity chromatography steps. After 40-80% ammonium sulfate fractionation, the enzyme was adsorbed to an N6-2′.5′-ADP-Sepharose affinity column from which it was specifically eluted by a 0 - 10 mᴍ NADP+ linear gradient. The enzyme was finally purified to homogeneity after a second affinity chromatography step in a C8-ATPR-Sepharose column, from which it was eluted by means of the same NADP+ gradient. Starting from 182 g of E. coli cells. 6.9 mg of pure enzyme was obtained after a 2632-fold purifi­cation, with a total yield of 63%. The pure enzyme showed a specific activity of 361 U/mg, and its absorption spectrum was characteristic of a flavoprotein. with an A272A450 of 7.84. The enzyme was a dimer with a molecular weight 109 000 and 40 Å hydrodynamic radius. The optimum pH were 7.5 and 4.5 with NADPH and NADH. respectively, as reductants. Apparent K′m values of 16, 377, and 66 μᴍ were determined at pH 7.5 for NADPH, NADH, and GSSG, respectively. Upon storage the enzyme was stable at pH values ranging from 7.5 to 9.5, being additionally stabilized by FAD. NADP+, dithiothreitol, or glycerol. The pure enzyme was quite heat stable, denaturing signifi­cantly only after 10 min at 70 0C. A marked activity loss was observed however, even at 0 °C, in the presence of 20 μᴍ NADPH. The enzyme was inactivated by low concentrations of para- hydroximercuribenzoate: the sensitivity towards such mercurial was greatly enhanced after reduction of the enzyme by NADPH.

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