Rapid Gene Cloning, Overexpression and Characterization of a Thermophilic Catalase in E. coli

2011 ◽  
Vol 365 ◽  
pp. 367-374 ◽  
Author(s):  
Hui Luo ◽  
Yao Zhou ◽  
Yan Hong Chang ◽  
Lian Xiong ◽  
Lin Zhi Liu
Keyword(s):  
Prokaryotic Expression ◽  
Bacillus Stearothermophilus ◽  
Base Pairs ◽  
Reading Frame ◽  
E Coli ◽  
Metal Affinity ◽  
Culture Optimization ◽  
Dna Mixture ◽  
Encoding Gene

A thermophilic catalase-encoding gene was rapidly obtained by means a PCR-based protocol with the genomic DNA mixture from compost culture as the template. The open reading frame of this gene is composed of 2208 base pairs, sharing 92.5% homology with the reported Bacillus stearothermophilus gene (NCBI genbank accession No. AB020234. 1). A prokaryotic expression plasmid pET-CATHis was constructed for the gene expression, and two recombinant E. coli, BL21(DE3)/pET-CATHis and BL21(DE3)pLysS/pET-CATHis were finally obtained. After culture optimization, the highest activities for these two strains in shaking flask culture were 74.3 U/ml and 1055.3 U/ml, respectively. The 6 His-tagged recombinant catalase was then purified by using immobilized metal affinity chromatography, and the properties of the purified protein were finally characterized.

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

scholarly journals Cloning and characterization of α-pinene synthase from Chamaecyparis formosensis Matsum

Holzforschung ◽  
2009 ◽  
Vol 63 (1) ◽  
Author(s):  
Fang-Hua Chu ◽  
Pei-Min Kuo ◽  
Yu-Rong Chen ◽  
Sheng-Yang Wang
Keyword(s):  
Major Product ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Geranyl Diphosphate ◽  
Reading Frame ◽  
E Coli ◽  
Phylogenetic Taxonomy ◽  
Chamaecyparis Formosensis ◽  
Pcr Method

AbstractAnalyzing the gene sequences of terpene synthase (TPS) may contribute to a better understanding of terpenes biosynthesis and evolution of phylogenetic taxonomy.Chamaecyparis formosensisis an endemic and precious conifer of Taiwan. To understand the biosynthesis mechanism of terpenes in this tree, a full length of putative mono-TPS, named asCf-Pin(GeneBank accession no. EU099434), was obtained by PCR method and RACE extension. TheCf-Pinhas an 1887-bp open reading frame and encodes 628 amino acids. To identify the function ofCf-Pin,the recombinant protein fromEscherichia coliwas incubated with geranyl diphosphate, produced one major product, the structure of which was elucidated. GC/MS analysis and matching of retention time and mass spectrum with authentic standards revealed that this product isα-pinene. This is the first report of cloning of a mono-TPS and functionally expressed inE. coliand which could be identified asα-pinene synthase from a Cupressaceae conifer.

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.

Cloning, overexpression and characterization of the Bacillus phytase encoding gene, YMNphyA, in E. coli

2013 ◽  
Vol 24 ◽  
pp. S95
Author(s):  
Melis Savasan Sogut ◽  
Abdulmecit Gokce ◽  
Yavuz Ozturk ◽  
Sevnur Mandaci ◽  
Ebru Cayir ◽  
...  
Keyword(s):  
E Coli ◽  
Encoding Gene

scholarly journals Cloning, expression and characterization of a mammalian Nudix hydrolase-like enzyme that cleaves the pyrophosphate bond of UDP-glucose

2003 ◽  
Vol 370 (2) ◽  
pp. 409-415 ◽  
Author(s):  
Toshihiro YAGI ◽  
Edurne BAROJA-FERNÁNDEZ ◽  
Ryuji YAMAMOTO ◽  
Francisco José MUÑOZ ◽  
Takashi AKAZAWA ◽  
...  
Keyword(s):  
Human Thyroid ◽  
Kinetic Properties ◽  
Nudix Hydrolase ◽  
Sequence Analyses ◽  
Nucleotide Sugar ◽  
E Coli ◽  
Nudix Hydrolases ◽  
Encoding Gene ◽  
Glycogen Biosynthesis

A distinct UDP-glucose (UDPG) pyrophosphatase (UGPPase, EC 3.6.1.45) has been characterized using pig kidney (Sus scrofa). This enzyme hydrolyses UDPG, the precursor molecule of numerous glycosylation reactions in animals, to produce glucose 1-phosphate (G1P) and UMP. Sequence analyses of the purified enzyme revealed that, similar to the case of a nucleotide-sugar hydrolase controlling the intracellular levels of ADP-glucose linked to glycogen biosynthesis in Escherichia coli [Moreno-Bruna, Baroja-Fernández, Muñoz, Bastarrica-Berasategui, Zandueta-Criado, Rodríguez-López, Lasa, Akazawa and Pozueta-Romero (2001) Proc. Natl. Acad. Sci. U.S.A. 98, 8128—8132], UGPPase appears to be a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated Nudix hydrolases. A complete cDNA of the UGPPase-encoding gene, designated UGPP, was isolated from a human thyroid cDNA library and expressed in E. coli. The resulting cells accumulated a protein that showed kinetic properties identical to those of pig UGPPase.

Molecular Cloning and Characterization of a cDNA Encoding a Transferrin Homolog from Bombyx mori

1999 ◽  
Vol 380 (12) ◽  
pp. 1455-1459 ◽  
Author(s):  
Eun Young Yun ◽  
Seok Woo Kang ◽  
Jae Sam Hwang ◽  
Tae Won Goo ◽  
Sang Hyun Kim ◽  
...  
Keyword(s):  
Bombyx Mori ◽  
Cdna Sequence ◽  
The Self ◽  
Open Reading Frame ◽  
Iron Binding ◽  
Defense System ◽  
Reading Frame ◽  
E Coli ◽  
Self Defense

Abstract We isolated a cDNA representing a message that was strongly induced by injection with E. coli in Bombyx mori. The 2160 bp cDNA has an open reading frame of 644 amino acids and the deduced product a predicted molecular mass of 71 kDa. The cDNA sequence shared high homology with the transferrins known so far, and its deduced peptide had unique features of transferrins, that is, sites of cystein residues and iron binding. We suggest that the B. mori transferrin plays an important role in the self-defense system.

scholarly journals Cloning and Expression of the algL Gene, Encoding the Azotobacter chroococcum Alginate Lyase: Purification and Characterization of the Enzyme

1999 ◽  
Vol 181 (5) ◽  
pp. 1409-1414 ◽  
Author(s):  
Ana Peciña ◽  
Alberto Pascual ◽  
Antonio Paneque
Keyword(s):  
Gene Sequence ◽  
Azotobacter Vinelandii ◽  
Alginate Lyase ◽  
Azotobacter Chroococcum ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Gene Encoding ◽  
Reading Frame ◽  
Encoding Gene

ABSTRACT The alginate lyase-encoding gene (algL) ofAzotobacter chroococcum was localized to a 3.1-kbEcoRI DNA fragment that revealed an open reading frame of 1,116 bp. This open reading frame encodes a protein of 42.98 kDa, in agreement with the value previously reported by us for this protein. The deduced protein has a potential N-terminal signal peptide that is consistent with its proposed periplasmic location. The analysis of the deduced amino acid sequence indicated that the gene sequence has a high homology (90% identity) to the Azotobacter vinelandii gene sequence, which has very recently been deposited in the GenBank database, and that it has 64% identity to the Pseudomonas aeruginosa gene sequence but that it has rather low homology (15 to 22% identity) to the gene sequences encoding alginate lyase in other bacteria. The A. chroococcum AlgL protein was overproduced in Escherichia coli and purified to electrophoretic homogeneity in a two-step chromatography procedure on hydroxyapatite and phenyl-Sepharose. The kinetic and molecular parameters of the recombinant alginate lyase are similar to those found for the native enzyme.

Characterization of the Specific DNA Nicking Activity of Restriction Endonuclease N.BstNBI

2000 ◽  
Vol 381 (11) ◽  
pp. 1123-1125 ◽  
Author(s):  
R.D. Morgan ◽  
C. Calvet ◽  
M. Demeter ◽  
R. Agra ◽  
H. Kong
Keyword(s):  
Restriction Endonuclease ◽  
Cleavage Site ◽  
Bacillus Stearothermophilus ◽  
Cleavage Sites ◽  
Recognition Sequence ◽  
Base Pairs ◽  
Nicking Endonuclease ◽  
Dna Nicking ◽  
Unique Restriction

AbstractN.BstNBI is a unique restriction endonuclease isolated fromBacillus stearothermophilus. We have characterized the recognition sequence and the cleavage site of N.BstNBI. Mapping of cleavage sites of N.BstNBI showed that it recognizes an asymmetric sequence, 5′ GAGTC 3′, and cleaves only on the top strand 4 base pairs away from its recognition sequence. To verify the nicking activity of N.BstNBI, we have constructed two plasmids containing a single recognition sequence (pNB1) or no recognition site (pNB0). When pNB1 and pNB0 were incubated with the enzyme, N.BstNBI nicked only the plasmid pNB1, suggesting that N.BstNBI is a specific nicking endonuclease.

Purification and Characterization of Recombinant sTRAIL Expressed in Escherichia coli

2004 ◽  
Vol 36 (2) ◽  
pp. 118-122 ◽  
Author(s):  
Xiao-Xia Xia ◽  
Ya-Ling Shen ◽  
Dong-Zhi Wei
Keyword(s):  
Inclusion Bodies ◽  
Tumor Cell Line ◽  
Single Step ◽  
Dilution Method ◽  
High Concentration ◽  
Purification And Characterization ◽  
E Coli ◽  
Metal Affinity ◽  
Soluble Trail

Abstract As a potential anti-tumor protein, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has drawn considerable attention. This report presented the purification and characterization of soluble TRAIL, expressed as inclusion bodies in E. coli. sTRAIL inclusion bodies were solubilized and refolded at a high concentration up to 0.9 g/L by a simple dilution method. Refolded protein was purified to electrophoretic homogeneity by a single-step immobilized metal affinity chromatography. The purified sTRAIL had a strong cytotoxic activity against human pancreatic tumor cell line 1990, with ED50 about 1.5 mg/L. Circular dichroism and fluorescence spectrum analysis showed that the refolded sTRAIL had a structure similar to that of native protein with β-sheet secondary structure. This efficient procedure of sTRAIL renaturation may be useful for the mass production of this therapeutically important protein.

scholarly journals Identification and Characterization of CAMP Cohemolysin as a Potential Virulence Factor of Riemerella anatipestifer

2002 ◽  
Vol 184 (7) ◽  
pp. 1932-1939 ◽  
Author(s):  
Karen C. Crasta ◽  
Kim-Lee Chua ◽  
Sumathi Subramaniam ◽  
Joachim Frey ◽  
Hilda Loh ◽  
...  
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chromosomal Dna ◽  
Pcr Cloning ◽  
Reading Frame ◽  
E Coli ◽  
Recombinant Plasmids ◽  
Riemerella Anatipestifer ◽  
Hydrolysis Of

ABSTRACT Riemerella anatipestifer is responsible for exudative septicemia in ducks. The genetic determinant of the CAMP cohemolysin, cam, from a strain of R. anatipestifer was cloned and expressed in Escherichia coli. Chromosomal DNA from serotype 19 strain 30/90 was used to construct a gene library in pBluescript II SK(−) vector in E. coli XL-1-Blue strain. The clones containing recombinant plasmids were screened for the CAMP reaction with Staphylococcus aureus. Those that showed cohemolysis were chosen for further analysis by sequencing. One of these clones, JFRA8, was subcloned to identify the smallest possible DNA fragment containing the CAMP cohemolysin determinant, which was located on a 3,566-bp BamHI-BstXI fragment which specified a 1,026-bp open reading frame. Clones containing recombinant plasmids carrying cam obtained by PCR cloning into E. coli M15 strain secreted an active CAMP cohemolysin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analyses confirmed that the recombinant strain expressed a protein with a molecular mass of 37 kDa and that strains from serotypes 1, 2, 3, 5, 6, and 19 expressed the cohemolysin. The deduced amino acid sequence showed high homology to those of O-sialoglycoprotein endopeptidases. Hydrolysis of radioiodinated glycophorin A confirmed that Cam is a sialoglycoprotease.

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