scholarly journals Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-beta-(1 6)-galactanase gene

2004 ◽  
Vol 377 (3) ◽  
pp. 749-755 ◽  
Author(s):  
Toshihisa KOTAKE ◽  
Satoshi KANEKO ◽  
Aya KUBOMOTO ◽  
Md. Ashraful HAQUE ◽  
Hideyuki KOBAYASHI ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Signal Sequence ◽  
Trichoderma Viride ◽  
Degree Of Polymerization ◽  
Glycoside Hydrolases ◽  
Cloning And Expression ◽  
Arabinogalactan Protein ◽  
Reading Frame ◽  
Cloned Gene

A gene encoding endo-β-(1→6)-galactanase from Trichoderma viride was cloned by reverse transcriptase–PCR and expressed in Escherichia coli. The gene contained an open reading frame consisting of 1437 bp (479 amino acids). The deduced amino acid sequence of the protein showed little similarity with other known glycoside hydrolases. A signal sequence (20 amino acids) was found at the N-terminal region of the protein and the molecular mass of the mature form was calculated to be 50.488 kDa. The gene product expressed in E. coli as a recombinant protein fused with thioredoxin and His6 tags had almost the same substrate specificity and mode of action as native enzyme purified from a commercial cellulase preparation of T. viride, i.e. recombinant enzyme endo-hydrolysed β-(1→6)-galacto-oligomers with a DP (degree of polymerization) higher than 3, and it could also hydrolyse α-l-arabinofuranosidase-treated arabinogalactan protein from radish. It produced β-(1→6)-galacto-oligomers ranging from DP 2 to at least 8 at the initial hydrolysis stage and galactose and β-(1→6)-galactobiose as the major products at the final reaction stage. These results indicate that the cloned gene encodes an endo-β-(1→6)-galactanase. As far as we know, this is the first time an endo-β-(1→6)-galactanase has been cloned.

scholarly journals Cloning and Expression of Mycobacterium tuberculosis and Mycobacterium leprae Dihydropteroate Synthase in Escherichia coli

1999 ◽  
Vol 181 (21) ◽  
pp. 6814-6821 ◽  
Author(s):  
Vanida Nopponpunth ◽  
Worachart Sirawaraporn ◽  
Patricia J. Greene ◽  
Daniel V. Santi
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Mycobacterium Tuberculosis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Mycobacterium Leprae ◽  
Open Reading Frame ◽  
Cloning And Expression ◽  
Reading Frame ◽  
Dihydropteroate Synthase

ABSTRACT The genes for dihydropteroate synthase of Mycobacterium tuberculosis and Mycobacterium leprae were isolated by hybridization with probes amplified from the genomic DNA libraries. DNA sequencing revealed an open reading frame of 840 bp encoding a protein of 280 amino acids for M. tuberculosisdihydropteroate synthase and an open reading frame of 852 bp encoding a protein of 284 amino acids for M. leprae dihydropteroate synthase. The dihydropteroate synthases were expressed under control of the T5 promoter in a dihydropteroate synthase-deficient strain ofEscherichia coli. Using three chromatography steps, we purified both M. tuberculosis and M. lepraedihydropteroate synthases to >98% homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed molecular masses of 29 kDa for M. tuberculosis dihydropteroate synthase and 30 kDa for M. leprae dihydropteroate synthase. Gel filtration of both enzymes showed a molecular mass of ca. 60 kDa, indicating that the native enzymes exist as dimers of two identical subunits. Steady-state kinetic parameters for dihydropteroate synthases from bothM. tuberculosis and M. leprae were determined. Representative sulfonamides and dapsone were potent inhibitors of the mycobacterial dihydropteroate synthases, but the antimycobacterial agent p-aminosalicylate, a putative dihydropteroate synthase inhibitor, was a poor inhibitor of the enzymes.

scholarly journals Sequence and structure of mtr, an amino acid transport gene of Neurospora crassa

Genome ◽  
1991 ◽  
Vol 34 (4) ◽  
pp. 644-651 ◽  
Author(s):  
Kenneth Koo ◽  
W. Dorsey Stuart
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Rna Binding ◽  
Signal Sequence ◽  
Average Length ◽  
Open Reading Frame ◽  
Mrna Transcript ◽  
S1 Nuclease ◽  
Reading Frame

The gene product of the mtr locus of Neurospora crassa is required for the transport of neutral aliphatic and aromatic amino acids via the N system. We have previously cloned three cosmids containing Neurospora DNA that complement the mtr-6(r) mutant allele. The cloned DNAs were tightly linked to restriction fragment length polymorphisms that flank the mtr locus. A 2.9-kbp fragment from one cosmid was subcloned and found to complement the mtr-6(r) allele. Here we report the sequence of the fragment that hybridized to a poly(A)+ mRNA transcript of about 2300 nucleotides. We have identified an 845-bp open reading frame (ORF) having a 59-bp intron as the potential mtr ORF. S1 nuclease analysis of the transcript confirmed the transcript size and the presence of the intron. A second open reading frame was found upstream in the same reading frame as the mtr ORF and appears to be present in the mRNA transcript. The mtr ORF is predicted to encode a 261 amino acid polypeptide with a molecular mass of 28 613 Da. The proposed polypeptide exhibits six potential α-helical transmembrane domains with an average length of 23 amino acids, does not have a signal sequence, and contains amino acid sequence homologous to an RNA binding motif.Key words: sequence, membranes, ribonucleoprotein.

scholarly journals Cloning and expression of a Leishmania donovani gene instructed by a peptide isolated from major histocompatibility complex class II molecules of infected macrophages.

1995 ◽  
Vol 182 (5) ◽  
pp. 1423-1433 ◽  
Author(s):  
A Campos-Neto ◽  
L Soong ◽  
J L Cordova ◽  
D Sant'Angelo ◽  
Y A Skeiky ◽  
...  
Keyword(s):  
Mhc Class Ii ◽  
Leishmania Donovani ◽  
Class Ii ◽  
Cloning And Expression ◽  
Diagnostic Tools ◽  
Reading Frame ◽  
Dna Fragment ◽  
Peptide Gene ◽  
Cloned Gene ◽  
Sense Primer

The studies reported here describe the isolation of peptides from MHC class II molecules of murine macrophages infected with Leishmania donovani, and the use of the derived peptide sequences to rescue the pathogen peptide donor protein. The isolation of the peptides was carried out by comparing the RP HPLC profile of peptides extracted from infected macrophages with the peptides extracted from noninfected cells. Several distinct HPLC peaks unique to infected macrophages were sequenced. One of the peptides that was not homologous to any known protein was used to instruct the designing of an oligonucleotide sense primer that was used in combination with an oligo dT nucleotide (anti-sense primer) to amplify by PCR a DNA fragment from L. donovani cDNA. The amplified DNA fragment was cloned and used as a probe to screen a L. donovani cDNA library. The cloned gene (Ld peptide gene) has an open reading frame of 525 bp and has no homology with any known protein/gene sequence. Northern blot analyses indicated that the Ld peptide/gene is broadly distributed and expressed among species of the Leishmania genus, in both the amastigote and promastigote life cycle forms. Using the pGEX 2T vector, the gene was expressed and the relationship of the purified recombinant protein with L. donovani was confirmed using both antibody and T cell responses from immunized or infected animals. The gene encodes a 23-kD molecule (Ldp 23) associated with the cell surface of L. donovani promastigotes. In addition, T cells purified from the lymph nodes of BALB/c mice immunized with L. donovani or infected with L. major, and from CBA/J mice infected with L. amazonensis were stimulated to proliferate by the recombinant Ldp 23 and produced high levels of IFN-gamma and no IL 4. This observation suggests that the Ldp 23 is an interesting parasite molecule for the studies concerning the host/parasite interaction because the Th1 pattern of cytokine response that it induces is correlated with resistance to Leishmania infections. These results clearly point to an alternative strategy for the purification of proteins useful for the development of both vaccines and immunological diagnostic tools not only against leishmaniasis but also for other diseases caused by intracellular pathogens.

The N-Terminal Signal Sequence and the Last 98 Amino Acids Are Not Essential for the Secretion of Bacillus sp. TS-23 α-Amylase in Escherichia coli

2001 ◽  
Vol 43 (3) ◽  
pp. 170-175 ◽  
Author(s):  
Huei-Fen Lo ◽  
Long-Liu Lin ◽  
Chien-Cheng Li ◽  
Wen-Hwei Hsu ◽  
Chen-Tien Chang
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Signal Sequence ◽  
Bacillus Sp

Molecular cloning and sequence analysis of putative chicken prolactin cDNA

1989 ◽  
Vol 2 (1) ◽  
pp. 21-30 ◽  
Author(s):  
M.C. Hanks ◽  
J.A. Alonzi ◽  
P.J. Sharp ◽  
H.M. Sang
Keyword(s):  
Amino Acids ◽  
Genomic Dna ◽  
Signal Sequence ◽  
Peptide Sequence ◽  
Mrna Levels ◽  
Reading Frame ◽  
Ring Dove ◽  
Prolactin Gene ◽  
Pituitary Glands ◽  
High Degree

ABSTRACT A cDNA library was prepared from mRNA isolated from anterior pituitary glands of incubating bantam hens, in which prolactin mRNA levels were predicted to be very high. Nine clones, representing abundant mRNA species, were identified and shown to contain homologous sequences. Two clones, of 871 bp and 580 bp, were analysed by DNA sequencing. The shorter clone was found to be a truncated cDNA product but otherwise identical to the longer clone. The 871 bp cDNA, PRL101, contains an open reading frame capable of encoding a polypeptide of 229 amino acids. This putative polypeptide has a high degree of homology to mammalian prolactins (approximately 70%), strongly suggesting that PRL101 encodes chicken preprolactin. The protein was predicted to have a 30 amino acid signal sequence which would be cleaved off to give a mature protein of 199 amino acids. The peptide sequence also had a 26% homology to chicken growth hormone, which is related to prolactin. This similarity confirms the conclusion that PRL101 is a chicken prolactin cDNA clone. An abundant mRNA of approximately 880 b was detected in poly(A)+ RNA from pituitary glands probed with PRL101. Analysis of chicken genomic DNA showed that there is one copy of the prolactin gene in the genome. PRL101 hybridized strongly to genomic DNA from closely related galliforms (quail and turkey) and less strongly to DNA from more distantly related species (duck and ring dove).

A β-Mannanase from Bacillus subtilis B36: Purification, Properties, Sequencing, Gene Cloning and Expression in Escherichia coli

2006 ◽  
Vol 61 (11-12) ◽  
pp. 840-846 ◽  
Author(s):  
Ya Nan Li ◽  
Kun Meng ◽  
Ya Ru Wang ◽  
Bin Yao
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Bacillus Subtilis ◽  
Specific Activity ◽  
Ph Value ◽  
High Specificity ◽  
Apparent Molecular Weight ◽  
Cloning And Expression ◽  
Gene Cloning And Expression ◽  
Reading Frame

Abstract MANB36, a secrete endo-β-1,4-D-mannanase produced by Bacillus subtilis B36, was puri­fied to homogeneity from a culture supernatant and characterized. The optimum pH value for the mannanase activity of MANB36 is 6.4 and the optimum temperature is 50 °C. The enzyme activity of MANB36 is remarkably thermostable at 60 °C and the specific activity of MANB36 is 927.84 U/mg. Metal cations (except Hg2+ and Ag+), EDTA and 2-mercaptoetha- nol (2-ME) have no effects on enzyme activity. This enzyme exhibits high specificity with the substituted galactomannan locust bean gum (LBG). The gene encoding for MANB36, manB36, was cloned by PCR and sequenced. manB36 contains a single open reading frame (ORF) consisting of 1104 bp that encodes a protein of 367 amino acids. The predicted mo­lecular weight of 38.13 kDa, calculated by the deduced protein of the gene manB36 without signal peptide, coincides with the apparent molecular weight of 38.0 kDa of the purified MANB36 estimated by SDS-PAGE. The mature protein of MANB36 has been expressed in Escherichia coli BL21 and the expressed mannanase has normal bioactivity.

scholarly journals Characterization of the Collagen-Binding S-Layer Protein CbsA of Lactobacillus crispatus

2000 ◽  
Vol 182 (22) ◽  
pp. 6440-6450 ◽  
Author(s):  
Jouko Sillanpää ◽  
Beatriz Martínez ◽  
Jenni Antikainen ◽  
Takahiro Toba ◽  
Nisse Kalkkinen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Type I Collagen ◽  
Signal Sequence ◽  
Type I ◽  
Binding Region ◽  
Sequence Comparisons ◽  
Collagen Binding ◽  
Reading Frame ◽  
Lactobacillus Crispatus ◽  
C Terminus

The cbsA gene of Lactobacillus crispatusstrain JCM 5810, encoding a protein that mediates adhesiveness to collagens, was characterized and expressed in Escherichia coli. The cbsA open reading frame encoded a signal sequence of 30 amino acids and a mature polypeptide of 410 amino acids with typical features of a bacterial S-layer protein. ThecbsA gene product was expressed as a His tag fusion protein, purified by affinity chromatography, and shown to bind solubilized as well as immobilized type I and IV collagens. Three otherLactobacillus S-layer proteins, SlpA, CbsB, and SlpnB, bound collagens only weakly, and sequence comparisons of CbsA with these S-layer proteins were used to select sites in cbsAwhere deletions and mutations were introduced. In addition, hybrid S-layer proteins that contained the N or the C terminus from CbsA, SlpA, or SlpnB as well as N- and C-terminally truncated peptides from CbsA were constructed by gene fusion. Analysis of these molecules revealed the major collagen-binding region within the N-terminal 287 residues and a weaker type I collagen-binding region in the C terminus of the CbsA molecule. The mutated or hybrid CbsA molecules and peptides that failed to polymerize into a periodic S-layer did not bind collagens, suggesting that the crystal structure with a regular array is optimal for expression of collagen binding by CbsA. Strain JCM 5810 was found to contain another S-layer gene termed cbsB that was 44% identical in sequence to cbsA. RNA analysis showed that cbsA, but not cbsB, was transcribed under laboratory conditions. S-layer-protein-expressing cells of strain JCM 5810 adhered to collagen-containing regions in the chicken colon, suggesting that CbsA-mediated collagen binding represents a true tissue adherence property of L. crispatus.

scholarly journals Cloning and Expression of Serratia Marcescens Prodigiosin Gene in Ecoli XL1blue

2021 ◽  
Vol 20 (2) ◽  
pp. 102-111
Author(s):  
Kumarss Amini ◽  
◽  
Maryam Akbari ◽  
Keyword(s):  
Escherichia Coli ◽  
Serratia Marcescens ◽  
Pharmaceutical Products ◽  
Cloning And Expression ◽  
Dust Particles ◽  
Negative Bacterium ◽  
Phylogenetic Studies ◽  
Close Relatives ◽  
Tract Infections ◽  
Cloned Gene

Background and Objectives: Serratia is a gram-negative bacterium. The pigmentation property of Serratia Marcescens is used as a marker of dust particles in the environment and in the hospital. Today biopigments are also widely used in the manufacture and production of pharmaceutical products. Prodigiosin is a promising drug due to its reported properties of antifungal immunosuppressive and anti-proliferative activities. In the present study, cloning of pig gene- isolated from Serratia Marcescens in Ecoli XL1blue was performed. Subjects and Methods 60 Samples were taken from clinical sources of patients hospitalized with urinary tract infections in Saveh Hospitals. Serratia Marcescens were identified and isolated by different tests. The pig gene was cloned by T-A cloning using PTG-19 vector into the Escherichia coli XL1blue as host. Expression of cloned gene in recombinant colonies was evaluated by Real time PCR. The phylogenetic tree was plotted using clustalX and Mega5 software Results Screening of samples identified 12 isolates of Serratia Marcescens from then 4 isolates had pig gene. Expression of Pig gene in Escherichia coli XL1blue was confirmed by Real-Tima PCR. As a result of phylogenetic studies, some close relatives of serratia have been identified as candidates for further studies Conclusion Serratia Marcescens can be considered as a rich source of pigments with many applications and can be used as indigenous strains to produce Prodigiosin.

scholarly journals Properties of recombinant trehalose synthase from Deinococcus radiodurans expressed in Escherichia coli.

2012 ◽  
Vol 59 (3) ◽  
Author(s):  
Paweł Filipkowski ◽  
Olga Pietrow ◽  
Anna Panek ◽  
Józef Synowiecki
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Affinity Chromatography ◽  
Molecular Mass ◽  
Deinococcus Radiodurans ◽  
Trehalose Synthase ◽  
Reading Frame ◽  
Metal Affinity ◽  
C Terminus

A trehalose synthase gene from Deinococcus radiodurans (DSMZ 20539) containing 1659 bp reading frame encoding 552 amino acids was amplified using PCR. The gene was finally ligated into pET30Ek/LIC vector and expressed after isopropyl β-d-thiogalactopyranoside induction in Escherichia coli (DE3) Rosetta pLysS. The recombinant trehalose synthase (DraTreS) containing a His(6)-tag at the C-terminus was purified by metal affinity chromatography and characterized. The expressed enzyme is a homodimer with molecular mass of 126.9 kDa and exhibits the highest activity of 11.35 U/mg at pH 7.6 and at 30°C. DraTreS activity was almost unchanged after 2 h preincubation at 45°C and pH 7.6, and retained about 56% of maximal value after 8 h incubation at 50°C. The DraTreS was strongly inhibited by Cu(2+), Hg(2+), Zn(2+), Al(3+) and 10 mM Tris. The K(m) value of maltose conversion was 290.7 mM.

scholarly journals Identification, Cloning, and Expression of the CAMP factor gene (cfa) of Group A Streptococci

1999 ◽  
Vol 67 (9) ◽  
pp. 4725-4731 ◽  
Author(s):  
Klaus Gase ◽  
Joseph J. Ferretti ◽  
Charles Primeaux ◽  
W. Michael McShan
Keyword(s):  
Escherichia Coli ◽  
Cloning And Expression ◽  
Group A Streptococci ◽  
Reading Frame ◽  
Factor Gene ◽  
Streptococcus Uberis ◽  
Camp Factor ◽  
Amino Acid Signal Peptide ◽  
Group A ◽  
Beta Toxin

ABSTRACT The CAMP reaction is a synergistic lysis of erythrocytes by the interaction of an extracellular protein (CAMP factor) produced by some streptococcal species with the Staphylococcus aureussphingomyelinase C (beta-toxin). Group A streptococci (GAS [Streptococcus pyogenes]) have been long considered CAMP negative, and this reaction commonly has been used to distinguish GAS from Streptococcus agalactiae. We here provide evidence that GAS possess this gene and produce an extracellular CAMP factor capable of participating in a positive CAMP reaction. The S. pyogenes CAMP factor is specified by a 774-bp open reading frame homologous to the CAMP factor genes from S. agalactiae andStreptococcus uberis. This gene, designatedcfa, was isolated on a 1,256-bp fragment and cloned inEscherichia coli. Recombinant clones of E. coliexpressing cfa secreted an active CAMP factor. The deduced 28.5-kDa protein encoded by cfa consists of 257 amino acids, with a predicted 28-amino-acid signal peptide. Thecfa gene is widely spread among GAS: 82 of 100 clinical GAS isolates produced a positive CAMP reaction. Of the CAMP-negative strains, 17 of the 18 GAS strains contained the cfa gene. Additionally, CAMP activity was detected in streptococci from serogroups C, M, P, R, and U. The cfa gene was cloned and actively expressed in Escherichia coli and gene fusions were made, placing the β-galactosidase gene (lacZ) under control of the cfa promoter. These cfapromoter-lacZ fusions were introduced into S. pyogenes via a bacteriophage-derived site-specific integration vector where they showed that the cfa gene has a strong promoter that may be subject to as-yet-unidentified regulatory factors. The results presented here, along with previous reports, indicate that the CAMP factor gene is fairly widespread among streptococci, being present at least in groups A, B, C, G, M, P, R, and U.

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