Overexpression of the ftsZ gene from Corynebacterium glutamicum (Brevibacterium lactofermentum) in Escherichia coli

2005 ◽  
Vol 51 (1) ◽  
pp. 85-89 ◽  
Author(s):  
María Pilar Honrubia-Marcos ◽  
Angelina Ramos ◽  
José A Gil
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Polyclonal Antibodies ◽  
T7 Promoter ◽  
Brevibacterium Lactofermentum ◽  
E Coli ◽  
Ftsz Protein ◽  
Pure Protein ◽  
Ftsz Gene ◽  
Dna Deletions

Our goal in this work was to overexpress the essential cell division FtsZ protein from Corynebacterium glutamicum (Brevibacterium lactofermentum) (FtsZCG) in Escherichia coli to produce anti-FtsZCG polyclonal antibodies. Previous results from our laboratory showed that ftsZCG was not expressed in E. coli in a sufficient amount to purify FtsZCG. However, when ftsZCG (without upstream sequences) was transcriptionally fused to the T7 promoter, different truncated FtsZCG proteins (28–32 kDa) were overexpressed in E. coli, and in all cases, stop codons were created because of DNA deletions or rearrangements. Nevertheless, we were able to overexpress and purify an N-terminally hexa-His-tagged FtsZCG from uninduced E. coli cells carrying a pET-28a(+) derivative, yielding about 5 mg of 98% pure protein per 100-mL culture.Key words: Brevibacterium lactofermentum, Corynebacterium glutamicum, FtsZ overexpresssion, hexa-His-tagged FtsZ.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

scholarly journals Rhizobium (Sinorhizobium)meliloti phn Genes: Characterization and Identification of Their Protein Products

1999 ◽  
Vol 181 (2) ◽  
pp. 389-395 ◽  
Author(s):  
George F. Parker ◽  
Timothy P. Higgins ◽  
Timothy Hawkes ◽  
Robert L. Robson
Keyword(s):  
Escherichia Coli ◽  
Sinorhizobium Meliloti ◽  
Insertional Mutagenesis ◽  
Biochemical Characterization ◽  
Polyclonal Antibodies ◽  
E Coli ◽  
New Information ◽  
Phosphorus Sources ◽  
Carbon Phosphorus Lyase

ABSTRACT In Escherichia coli, the phn operon encodes proteins responsible for the uptake and breakdown of phosphonates. The C-P (carbon-phosphorus) lyase enzyme encoded by this operon which catalyzes the cleavage of C-P bonds in phosphonates has been recalcitrant to biochemical characterization. To advance the understanding of this enzyme, we have cloned DNA fromRhizobium (Sinorhizobium) melilotithat contains homologues of the E. coli phnG, -H, -I, -J, and -Kgenes. We demonstrated by insertional mutagenesis that the operon from which this DNA is derived encodes the R. meliloti C-P lyase. Furthermore, the phenotype of this phn mutant shows that the C-P lyase has a broad substrate specificity and that the organism has another enzyme that degrades aminoethylphosphonate. A comparison of the R. meliloti and E. coli phngenes and their predicted products gave new information about C-P lyase. The putative R. meliloti PhnG, PhnH, and PhnK proteins were overexpressed and used to make polyclonal antibodies. Proteins of the correct molecular weight that react with these antibodies are expressed by R. meliloti grown with phosphonates as sole phosphorus sources. This is the first in vivo demonstration of the existence of these hitherto hypothetical Phn proteins.

Analysis of p53 expression in human tumours: an antibody raised against human p53 expressed in Escherichia coli

1992 ◽  
Vol 101 (1) ◽  
pp. 183-189 ◽  
Author(s):  
C.A. Midgley ◽  
C.J. Fisher ◽  
J. Bartek ◽  
B. Vojtesek ◽  
D. Lane ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Polyclonal Antibodies ◽  
P53 Protein ◽  
Expression System ◽  
High Titre ◽  
T7 Promoter ◽  
Aberrant Expression ◽  
P53 Expression ◽  
Normal Human ◽  
Very High

A cDNA encoding the complete normal human p53 protein was expressed in Escherichia coli using an expression system based on the bacteriophage T7 promoter. The cDNA was adapted so that the full-length protein was produced without fusion to any other sequence. Large amounts of the protein were isolated and the purified protein used to produce very high titre polyclonal antibodies to p53. These new antibodies permit the sensitive detection of p53 and p53 complexes in ELISA and immunoblotting assays. Most importantly, they also permit the detection of p53 in archival tumour material that has been conventionally fixed in formalin and embedded in paraffin wax. Using this reagent we have found that aberrant expression of p53 is a frequent feature of human breast cancer. We are able to recognise six different classes of p53 expression pattern that may be of help in the subclassification of breast tumours.

Co-Expression of Threonine Dehydratase and Acetolactate Synthase in Escherichia Coli for L-Isoleucine Production

2014 ◽  
Vol 989-994 ◽  
pp. 997-1002 ◽  
Author(s):  
Jian Wang ◽  
Jia Kai Sun ◽  
Qing Yang Xu
Keyword(s):  
Escherichia Coli ◽  
Corynebacterium Glutamicum ◽  
Carbon Flux ◽  
Batch Fermentation ◽  
Acetolactate Synthase ◽  
Fed Batch ◽  
Threonine Dehydratase ◽  
E Coli ◽  
Fed Batch Fermentation ◽  
Fermentation Period

Metabolic engineering ofCorynebacterium glutamicumhas sought to divert carbon into L-isoleucine. However, the fermentation period of this strain is long. TheC.glutamicumYILW strain (LeuL, AHVr, SGr, Leu-MEr) was previously derived by repeated compound mutagenesis which could accumulate 20.2 g/L L-isoleucine in a 5-L jar fermentor. Overexpression of the threonine dehydratase gene (ilvA) fromCorynebacterium glutamicumYILW and coexpression of threonine dehydratase and acetolactate synthase (ilvBN) from it were employed to divert carbon flux toward L-isoleucine. The strainE. coliTRFC with the expression ofilvA could accumulate L-isoleucine of 6.8 g/L without accumulation of any L-threonine by fed-batch fermentation in a 5-L jar fermentor. However, the production of L-isoleucine by the strainE.coliTRFC with the co-expression ofilvA andilvBN was decreased by 19.1%, and the production of L-valine was increased by 40% compared with that ofE. coliTRFC with the expression ofilvA.

Cloning and expression of a functional rat liver D-β-hydroxybutyrate dehydrogenase-β-galactosidase fusion protein in Escherichia coli

1991 ◽  
Vol 69 (9) ◽  
pp. 670-673
Author(s):  
Sharon Churchill ◽  
Perry Churchill
Keyword(s):  
Escherichia Coli ◽  
Rat Liver ◽  
Polyclonal Antibodies ◽  
Cloning And Expression ◽  
Dependent Manner ◽  
Bacteriophage Λ ◽  
Expression Library ◽  
Expression Plasmid ◽  
E Coli ◽  
Dose Dependent

A rat liver bacteriophage λ expression library was probed using polyclonal antibodies raised to purified rat liver D-β-hydroxybutyrate dehydrogenase (BDH). A clone was selected that contained a 1.2-kb insert. The insert placed in an expression plasmid was utilized to transform Escherichia coli. These cells were shown to possess phosphatidylcholine-dependent BDH activity. Cells transformed with only the plasmid had no detectable BDH activity in the presence of phosphatidylcholine. The expressed activity in E. coli could be inhibited in a dose-dependent manner by BDH antiserum.Key words: D-β-hydroxybutyrate dehydrogenase, cloning, expression.

scholarly journals Impact of Heterologous Expression of Escherichia coli UDP-Glucose Pyrophosphorylase on Trehalose and Glycogen Synthesis in Corynebacterium glutamicum

2004 ◽  
Vol 70 (7) ◽  
pp. 3845-3854 ◽  
Author(s):  
Leandro Padilla ◽  
Susanne Morbach ◽  
Reinhard Krämer ◽  
Eduardo Agosin
Keyword(s):  
Escherichia Coli ◽  
Heterologous Expression ◽  
Corynebacterium Glutamicum ◽  
Glycogen Synthesis ◽  
Glycogen Accumulation ◽  
E Coli ◽  
Wide Range ◽  
The Impact ◽  
Synthetic Operon ◽  
Trehalose Synthesis

ABSTRACT Trehalose is a disaccharide with a wide range of applications in the food industry. We recently proposed a strategy for trehalose production based on improved strains of the gram-positive bacterium Corynebacterium glutamicum. This microorganism synthesizes trehalose through two major pathways, OtsBA and TreYZ, by using UDP-glucose and ADP-glucose, respectively, as the glucosyl donors. In this paper we describe improvement of the UDP-glucose supply through heterologous expression in C. glutamicum of the UDP-glucose pyrophosphorylase gene from Escherichia coli, either expressed alone or coexpressed with the E. coli ots genes (galU otsBA synthetic operon). The impact of such expression on trehalose accumulation and excretion, glycogen accumulation, and the growth pattern of new recombinant strains is described. Expression of the galU otsBA synthetic operon resulted in a sixfold increase in the accumulated and excreted trehalose relative to that in a wild-type strain. Surprisingly, single expression of galU also resulted in an increase in the accumulated trehalose. This increase in trehalose synthesis was abolished upon deletion of the TreYZ pathway. These results proved that UDP-glucose has an important role not only in the OtsBA pathway but also in the TreYZ pathway.

scholarly journals Analysis of Bioavailable Toluene by Using Recombinant Luminescent Bacterial Biosensors with Different Promoters

2020 ◽  
Author(s):  
Guey-Horng Wang ◽  
Teh-Hua Tsai ◽  
Chun-Chi Kui ◽  
Chiu-Yu Cheng ◽  
Tzu-Ling Huang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Organic Compounds ◽  
Luminescence Intensity ◽  
Limit Of Detection ◽  
Measurement Range ◽  
T7 Promoter ◽  
E Coli ◽  
Low Concentrations ◽  
Relative Standard ◽  
Acceptable Deviation

Abstract In this study, we constructed recombinant luminescent Escherichia coli with T7, T3, and SP6 promoters inserted between tol and lux genes as toluene biosensors and evaluated their sensitivity, selectivity, and specificity for measuring bioavailable toluene in in groundwater and river water. The luminescence intensity of each biosensor depended on temperature, incubation time, ionic strength, and concentrations of toluene and coexisting organic compounds. Toluene induced the highest luminescence intensity in recombinant lux-expressing E. coli with the T7 promoter [T7-lux-E. coli, limit of detection (LOD) = 0.05 μM], followed by that in E. coli with the T3 promoter (T3-lux-E. coli, LOD = 0.2 μM) and SP6 promoter (SP6-lux-E. coli, LOD = 0.5 μM). Luminescence activities may have been synergistically or antagonistically affected by coexisting organic compounds other than toluene; nevertheless, low concentrations of benzoate and toluene analogs had no such effect. In reproducibility experiments, the biosensors had low relative standard deviation (4.3%–5.8%). SP6-lux-E. coli demonstrated high adaptability to environmental interference. T7-lux-E. coli biosensor—with low LOD, wide measurement range (0.05–500 μM), and acceptable deviation (−14.3% to 9.1%)—is an efficient toluene biosensor. This is the first study evaluating recombinant lux E. coli with different promoters for their potential application in toluene measurement in actual water bodies.

scholarly journals Ferulic Acid production by metabolically engineered Escherichia coli

2021 ◽  
Author(s):  
Huajun Lv ◽  
Ying Zhang ◽  
Jie Shao ◽  
Haili Liu ◽  
Yong Wang
Keyword(s):  
Escherichia Coli ◽  
Ferulic Acid ◽  
Caffeic Acid ◽  
Pyridine Nucleotide ◽  
Promoter Strength ◽  
T7 Promoter ◽  
Nadph Regeneration ◽  
E Coli ◽  
Ferulic Acid Production ◽  
K 12

Abstract Ferulic acid (p-hydroxy-3-methoxycinnamic acid, FA) is a natural active substance present in plant cell walls, with antioxidant, anticancer, antithrombotic and other properties; it is widely used in medicine, food, and cosmetics areas. Production of FA by eco-friendly bioprocess is of great potential. In this study, FA was biosynthesized by metabolically engineered Escherichia coli. As the first step, the genes tal (encoding Tyrosine ammonia-lyase, RsTAL) from Rhodobacter sphaeroides, sam5 (encoding p - coumarate 3-hydroxylase, SeSAM5) from Saccharothrix espanaensis and comt (encoding Caffeic acid O-methytransferase, TaCM) from Triticum aestivum were cloned in an operon on the pET plasmid backbone, E. coli strain containing this construction was proved to produce FA from L-tyrosine successfully, and confirmed the function of TaCM as Caffeic acid O-methytransferase. Fermentation results revealed JM109(DE3) as more suitable host cell for FA production than BL21(DE3). After that the genes expression strength of FA pathway were optimized by tuning of promoter strength (T7 promoter or T5 promoter) and copy number (pBR322 ori or p15a ori), and the combination p15a-T5 works best. To further improve FA production, E.coli native pntAB, encoding pyridine nucleotide transhydrogenase, was selected from five NADPH regeneration genes to supplement redox cofactor NADPH for converting p-coumaric acid into caffeic acid in FA biosynthesis process. Sequentially, to further convert caffeic acid into FA, a non-native methionine kinase (MetK from Streptomyces spectabilis) was also over expressed. Based on the flask fermentation data which shows that the engineered E. coli strain produced 212 mg/L of FA with 11.8 mg/L caffeic acid residue, it could be concluded that it is the highest yield of FA achieved by E.coli K-12 strains reported to the best of our knowledge.

Implementation of a novel self-induced promoter for the expression of pharmaceutical peptides in Escherichia coli: YY(3-36) peptide

2019 ◽  
Vol 0 (0) ◽  
Author(s):  
Amir Hossein Momen ◽  
Naser Harzandi ◽  
Azam Haddadi ◽  
Bijan Bambai
Keyword(s):  
Escherichia Coli ◽  
Large Scale ◽  
Peptide Yy ◽  
Signal Sequence ◽  
Expression System ◽  
Promoter Sequence ◽  
Scale Production ◽  
Signal Sequences ◽  
T7 Promoter ◽  
E Coli

Abstract Background Increasing the expression rate of recombinant mammalian hormones in Escherichia coli by combining efficient promoters and signal sequences is a never ending process. A self-induced promoter will have some beneficial gains compared to the classical T7 promoter or its variants with isopropyl β-D-1-thiogalactopyranoside (IPTG) as the inducer. Obesity is the prime suspect in widespread frequency of diabetes type II and cardiovascular diseases worldwide. YY (tyrosine-tyrosine) peptide is a local acting hormone, controlling appetite. Excitingly, it was has been shown that a truncated version of the YY peptide, YY(3-36) peptide, has potential as a worthy biopharmaceutical agent in the fight against obesity. Materials and methods To develop an economical expression system for the large scale production of the peptide in Gram-negative bacteria, we introduced a promoter sequence upstream of a chimeric gene for the extracellular expression of this peptide with the assistance of a signal sequence of asparaginase II from E. coli. This system has the advantage of producing a complete sequence of a truncated YY peptide, YY(3-36), without any extra tags that would require further removal with the assistance of expensive specific proteases and reduced the downstream steps, significantly. Results Recombinant production of YY(3-36) peptide under a self-induced promoter proves the efficacy of the asparaginase II signal sequence as a communicator of foreign peptides and proteins into the extracellular space of E. coli. Conclusions The application of fusion protein expression of biopharmaceuticals, especially mammalian hormones, in prokaryotic systems with the help of native signal sequences makes some common tags with expensive proteases for the removal of the attached protein Tag redundant.

scholarly journals Characterization of a mammalian homolog of the Escherichia coli MutY mismatch repair protein.

1995 ◽  
Vol 15 (2) ◽  
pp. 989-996 ◽  
Author(s):  
J P McGoldrick ◽  
Y C Yeh ◽  
M Solomon ◽  
J M Essigmann ◽  
A L Lu
Keyword(s):  
Escherichia Coli ◽  
Specific Binding ◽  
Polyclonal Antibodies ◽  
Gradient Centrifugation ◽  
Immunoblot Analysis ◽  
Partial Purification ◽  
Phosphodiester Bond ◽  
E Coli ◽  
Nuclear Extracts ◽  
Calf Thymus

A protein homologous to the Escherichia coli MutY protein, referred to as MYH, has been identified in nuclear extracts of calf thymus and human HeLa cells. Western blot (immunoblot) analysis using polyclonal antibodies to the E. coli MutY protein detected a protein of 65 kDa in both extracts. Partial purification of MYH from calf thymus cells revealed a 65-kDa protein as well as a functional but apparently degraded form of 36 kDa, as determined by glycerol gradient centrifugation and immunoblotting with anti-MutY antibodies. Calf MYH is a DNA glycosylase that specifically removes mispaired adenines from A/G, A/7,8-dihydro-8-oxodeoxyguanine (8-oxoG or GO), and A/C mismatches (mismatches indicated by slashes). A nicking activity that is either associated with or copurified with MYH was also detected. Nicking was observed at the first phosphodiester bond 3' to the apurinic or apyrimidinic (AP) site generated by the glycosylase activity. The nicking activity on A/C mismatches was 30-fold lower and the activity on A/GO mismatches was twofold lower than that on A/G mismatches. No nicking activity was detected on substrates containing other selected mismatches or homoduplexes. Nicking activity on DNA containing A/G mismatches was inhibited in the presence of anti-MutY antibodies or upon treatment with potassium ferricyanide, which oxidizes iron-sulfur clusters. Gel shift analysis showed specific binding complex formation with A/G and A/GO substrates, but not with A/A, C.GO, and C.G substrates. Binding is sevenfold greater on A/GO substrates than on A/G substrates. The eukaryotic MYH may be involved in the major repair of both replication errors and oxidative damage to DNA, the same functions as those of the E. coli MutY protein.

Sign in / Sign up

Export Citation Format

Share Document