scholarly journals Identification of a New Class of Cytochrome P450 from a Rhodococcus sp

2002 ◽  
Vol 184 (14) ◽  
pp. 3898-3908 ◽  
Author(s):  
Gareth A. Roberts ◽  
Gideon Grogan ◽  
Andy Greter ◽  
Sabine L. Flitsch ◽  
Nicholas J. Turner
Keyword(s):  
Cytochrome P450 ◽  
Cytochromes P450 ◽  
Pcr Primers ◽  
Soluble Form ◽  
Flavin Mononucleotide ◽  
Gene Encoding ◽  
Binding Motifs ◽  
E Coli ◽  
New Class ◽  
C Terminus

ABSTRACT A degenerate set of PCR primers were used to clone a gene encoding a cytochrome P450 (the P450RhF gene) from Rhodococcus sp. strain NCIMB 9784 which is of unique primary structural organization. Surprisingly, analysis of the translation product revealed that the P450 is fused to a reductase domain at the C terminus which displays sequence conservation for dioxygenase reductase proteins. The reductase partner comprises flavin mononucleotide- and NADH-binding motifs and a [2Fe2S] ferredoxin-like center. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a soluble form. A recombinant strain of E. coli was able to mediate the O dealkylation of 7-ethoxycoumarin in good yield, despite the absence of any recombinant redox proteins. This unprecedented finding leads us to propose that P450RhF represents the first example of a new class of cytochromes P450 in which the reducing equivalents are supplied by a novel reductase in a fused arrangement.

scholarly journals Cloning, expression, purification and interaction evaluation of 30 amino acids in C terminus of Clostridium perfringens toxin with its receptor Claudin-4

2019 ◽  
Vol 2 (4) ◽  
pp. 47-55
Author(s):  
Quang Kien Huynh ◽  
An Hoang Nguyen ◽  
Quynh Thi Mong Pham ◽  
Hoan Phuoc Khai Nguyen ◽  
Hieu Van Tran
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Clostridium Perfringens ◽  
Field Effect Transistors ◽  
Oral Vaccine ◽  
Soluble Form ◽  
M Cells ◽  
Gastrointestinal Infections ◽  
E Coli ◽  
C Terminus

Oral vaccine is a strategy being the most interested about treatments of gastrointestinal infections because of many great benefits outweigh conventional injection vaccines. In order to resolve the dispersion of antigens in gastrointestinal surfaces, the immunological tolerance and also be capable to stimulate immune responses effectively, M cells are targeted for antigens delivery. A number of researches reported that 30 amino acids in C terminus of Clostridium perfringens toxin (CPE30) have a high affinity to Claudin-4 receptor presenting on M cells. It is highly indispensable to produce a resource for assessing of CPE30 binding ability so cpe30 gene was cloned into the pET-gfp plasmid by two restriction enzymes BamHI and NdeI on the E. coli DH5α strain. The expression and confirmation of the fusion protein CPE30-GFP which was induced by IPTG in E. coli BL21 (DE3) strain and assessed by SDS-PAGE and Western blot with 6xHis Taq antibody demonstrated that there was the over expression of CPE30 GFP fusion protein in the cytoplasm, mainly in the soluble form. Finally, CPE30-GFP was purified which the purity was approximately 92.3%. In vitro protein interaction measurement using silicon nanowire field-effect transistors (SiNW FETs) showed that CPE30-GFP had a good binding affinity with its receptor Claudin-4 (R4). This result laid the groundwork for the CPE30 interaction study with the M cell in vivo.

scholarly journals Expression of gene encoding flavonol synthase isolating from trung du xanh tea (Camellia sinensis var. macrophylla) in E. coli

2020 ◽  
Vol 42 (1) ◽  
Author(s):  
Hoang Thi Thu Yen ◽  
Vu Thi Lan ◽  
Huynh Thi Thu Hue
Keyword(s):  
Camellia Sinensis ◽  
Expression Vector ◽  
Soluble Fraction ◽  
Soluble Form ◽  
Gene Encoding ◽  
Flavonol Synthase ◽  
E Coli ◽  
Insoluble Form

Common flavonols in plants including quercetin, kaempferol and myricetin are synthesized from dihydroflavonols (dihydroquercetin-DHQ, dihydrokaempferol-DHK and dihydromyricetin-DHM) by flavonol synthase (FLS). In tea, FLS has been shown to metabolize dihydroquercetin to quercetin. The FLS gene was cloned and sequenced from the cultivated tea (Camellia sinensis var. macrophylla) in Thai Nguyen province. In this study, we presented the results of optimizing and designing an expression vector for recombinant FLS (recombinant FLS-rFLS). The FLS gene was ligated completely to the pET32a (+) vector, then expressed in E. coli Rosetta1 and Rosetta2 strain. Using 1mM IPTG to induce the expression of rFLS at 37oC, rFLS was obtained with 52.83 kDa in size and existed predominantly as insoluble form. E. coli Rosetta1 pET32a (+)_FLSproduces rFLS in the soluble fraction than E. coli Rosetta2 pET32a (+)_FLS. Next, E. coli Rosetta1 pET32a (+)_FLSwas optimized for expression at temperatures of 30oC, 23oC and 16oC (24 and 48 hours). After being induced for expression with 1mM IPTG in 48 hours and cultured at 16oC, E. coli Rosetta1 strain containing pET32a (+) FLS produced the largest amount of rFLS in the soluble form. 

scholarly journals Expression of Overlapping PreS1 Fragment Recombinant Proteins for the Determination of Immunogenic Domains in HBsAg PreS1 Region

2004 ◽  
Vol 36 (6) ◽  
pp. 397-404 ◽  
Author(s):  
Wei-Guo Hu ◽  
Jun Wei ◽  
Xin-Xiu Yang ◽  
Heng-Chuan Xia ◽  
Feng Li ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
Fusion Proteins ◽  
Competitive Elisa ◽  
Soluble Form ◽  
Immunological Property ◽  
E Coli ◽  
C Terminus ◽  
New Vaccines ◽  
Gst Fusion Proteins

Abstract In this study, eight preS1 fragments overlapped in preS1 (21–119) region of HBV adr subtype, i.e. preS1 (21–47), preS1 (34–59), preS1 (48–70), preS1 (60–85), preS1 (71–94), preS1 (86–109), preS1 (95–119) and preS1 (21–119), were cloned by PCR, and expressed as GST fusion proteins. These GST-preS1 fusion proteins were highly expressed in soluble form in E. coli, and about 50 to 90 mg soluble fusion proteins were purified from 1 L culture. Using these fusion proteins, the immunogenic domains in preS1 (21–119) region were identified by Western blot analysis and competitive ELISA. The results showed that the immunogenic domains mainly existed in preS1 (21–59) in N-terminus and preS1 (95–109) in C-terminus, and more importantly, a major immunogenic domain preS1 (34–59), which has much stronger immunogenicity, was identified. It was also supported by the predictions of secondary structure and immunological property in the preS1 (21–119) region. The results here would be helpful for the design of new vaccines against HBV.

Purification and characterization ofThermobifida fuscaxylanase 10B

2004 ◽  
Vol 50 (10) ◽  
pp. 835-843 ◽  
Author(s):  
Jeong H Kim ◽  
Diana Irwin ◽  
David B Wilson
Keyword(s):  
Enzyme Purification ◽  
Major Product ◽  
Corn Fiber ◽  
Thermobifida Fusca ◽  
Purification And Characterization ◽  
Gene Encoding ◽  
E Coli ◽  
Pure Cellulose ◽  
C Terminus ◽  
Xylanase Enzyme

Thermobifida fusca grows well on cellulose and xylan, and produces a number of cellulases and xylanases. The gene encoding a previously unstudied endoxylanase, xyl10B, was overexpressed in E. coli, and the protein was purified and characterized. Mature Xyl10B is a 43-kDa glycohydrolase with a short basic domain at the C-terminus. It has moderate thermostability, maintaining 50% of its activity after incubation for 16 h at 62 °C, and is most active between pH 5 and 8. Xyl10B is produced by growth of T. fusca on xylan or Solka Floc but not on pure cellulose. Mass spectroscopic analysis showed that Xyl10B produces xylobiose as the major product from birchwood and oat spelts xylan and that its hydrolysis products differ from those of T. fusca Xyl11A. Xyl10B hydrolyzes various p-nitrophenyl-sugars, including p-nitrophenyl α-D-arabinofuranoside, p-nitrophenyl-β-D-xylobioside, p-nitrophenyl-β-D-xyloside, and p-nitrophenyl-β-D-cellobioside. Xyl11A has higher activity on xylan substrates, but Xyl10B produced more reducing sugars from corn fiber than did Xyl11A.Key words: xylanase, enzyme purification, Thermobifida fusca, family 10 hydrolase.

Gene cloning, protein purification, and enzymatic properties of multicopper oxidase, fromKlebsiellasp. 601

2008 ◽  
Vol 54 (9) ◽  
pp. 725-733 ◽  
Author(s):  
Yang Li ◽  
Jiao Yin ◽  
Guosheng Qu ◽  
Luchao Lv ◽  
Yadong Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Kinetic Studies ◽  
Multicopper Oxidase ◽  
Coli Strain ◽  
Gene Encoding ◽  
E Coli ◽  
C Terminus ◽  
Optimal Ph

A gene encoding a putative multicopper oxidase (MCO) was cloned from the soil bacterium Klebsiella sp. 601 and its corresponding enzyme was overexpressed in an Escherichia coli strain. Klebsiella sp. 601 MCO is composed of 536 amino acids with a molecular mass of 58.2 kDa. Theoretical calculation gave a pI value of 6.11. The amino acid sequence of Klebsiella sp. 601 MCO is strongly homologous to that of E. coli CueO with a similarity of 90% and an identity of 78%. Unlike E. coli CueO, Klebsiella sp. 601 MCO contains an extra 20 amino acids close to its C-terminus. The enzyme was purified to homogeneity by Ni-affinity chromatography. The purified enzyme was capable of using DMP (2,6-dimethoxyphenol), ABTS (2,2′-azino-bis(3-ethylbenzthiazolinesulfonic acid)), and SGZ (syringaldazine) as substrates with an optimal pH of 8.0 for DMP, 3.0 for ABTS, and 7.0 for SGZ. Klebsiella sp. 601 MCO was quite stable at pH 7.0 in which its activity was constant for 25 h without any significant change. Kinetic studies gave Km, kcat, and kcat/Kmvalues of 0.49 mmol·L–1, 1.08 × 103s–1, and 2.23 × 103s–1·mmol–1·L, respectively, for DMP, 5.63 mmol·L–1, 6.64 × 103s–1, and 1.18 × 103s–1·mmol–1·L for ABTS, and 0.023 mmol·L–1, 11 s–1, and 4.68 × 102s–1·mmol–1·L for SGZ.

Heterologous expression and characterization of flavinadenine dinucleotide synthetase from Candida famata for flavin adenine dinucleotide production

2020 ◽  
Vol 27 ◽  
Author(s):  
Guoqiang Zhou ◽  
Qiaoqiao Pan ◽  
Zeyu Hu ◽  
Juanping Qiu ◽  
Zhiliang Yu
Keyword(s):  
Flavin Adenine Dinucleotide ◽  
Flavin Mononucleotide ◽  
Enzymatic Reactions ◽  
Gene Encoding ◽  
Food Industries ◽  
E Coli ◽  
Redox Active ◽  
Candida Famata ◽  
Optimized Conditions

Background: Flavin adenine dinucleotide (FAD) is a redox-active coenzyme that regulates several important enzymatic reactions during metabolism. FAD is used in the medicinal and food industries and FAD supplements have been used to treat some inheritable diseases. FAD can be biosynthesized from flavin mononucleotide (FMN) and adenosine triphosphate (ATP), catalyzed by FAD synthetase (FADS). Objective: The aim of this study was to heterologously express the gene encoding FADS from the flavinogenic yeast Candida famata (FADSCf) for biosynthesis of FAD. Methods: The sequence encoding FADSCf was retrieved and heterologously expressed in Escherichia coli. The structure and enzymatic properties of recombinant FADSCf were characterized. Results: FADSCf (279 amino acids) was successfully expressed in E. coli BL21 (DE3), with a theoretical molecular weight of 32299.79 Da and an isoelectric point of 6.09. Secondary structural analysis showed that the number of α-helices was 2-fold higher than the number of β-sheets, indicating that the protein was highly hydrophilic. Under fixed ATP concentration, FADSCf had a Km of 0.04737±0.03158 mM and a Vmax of 3.271±0.79 μM/min/mg. Under fixed FMN concentration, FADSCf had a Km of 0.1214±0.07464 mM and a Vmax of 2.6695±0.3715 μM/min/mg. Enzymatic reactions in vitro showed that expressed FADSCf could form 80 mM of FAD per mg of enzyme after 21 hours under the following conditions: 0.5 mM FMN, 5 mM ATP and 10 mM Mg2+ . Conclusion: Under optimized conditions (0.5 mM FMN, 5 mM ATP and 10 mM Mg2+), the production of FAD reached 80 mM per mg of FADSCf after a 21-hour reaction. Our results indicate that purified recombinant FADSCf can be used for the biosynthesis of FAD.

scholarly journals Probe design for exploiting gene encoding pectinesterase from dna metagenome data of bacteria in goat rumen and co-expression of gpecs1 gen with chaperone pg-kje8 in Escherichia coli

2018 ◽  
Vol 40 (1) ◽  
pp. 84-91
Author(s):  
Nguyen Khanh Hoang Viet ◽  
Do Thi Huyen ◽  
Le Tung Lam ◽  
Phung Thi Lan ◽  
Phung Thu Nguyet ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Dna Sequences ◽  
Soluble Form ◽  
Probe Design ◽  
Sequencing Data ◽  
Gene Encoding ◽  
E Coli ◽  
Environmental Treatment ◽  
Query Coverage ◽  
Goat Rumen

This article introduces the steps of constructing and using probe to exploit the gene encoding pectinesterase from metagenome DNA sequencing data by next generation gene sequencing tools. Probe was used to exploit and select the gene encoding for pectinesterase from the metagenome DNA sequences of bacteria in goat rumen and thereby select a sequence to express in E. coli. According to the CAZy classification system, pectinesterase belongs to the family of carbohydrates esterases CE8 is an enzyme that has many applications in the food processing industry, environmental treatment, animal feed processing and medicine. As the results, 3 sequences of CE8 was retrieved from CAZy database and one probe was designed, this probe length was 367 amino acids contained all the conserved amino acid residues: 200 conserved residues in all sequence, 72 residues similar in almost sequences and residues conserved in many sequences and homologus; choosed highest alkalinity index. Using the probe designed, we filtered four coding sequences for pectinesterase from metagenome DNA sequencing data of bacteria in goat rumen. Spatial structure estimation with Phyre2 has only one sequencing (code 46301) with 100% sequence identity and 90% query coverage with pectinesterase. A artificial gene were synthesized and inserted into the vector pET22b (+) at the NcoI, XhoI to co-express with chaperone pG-KJE8 in E. coli. The recombinant pectinesterase enzyme is expressed in soluble form and has a pectin substrate biodegradation activity. The results demonstrate that using probe for gene extraction is feasible.

scholarly journals Vibrio cholerae FabV Defines a New Class of Enoyl-Acyl Carrier Protein Reductase

2007 ◽  
Vol 283 (3) ◽  
pp. 1308-1316 ◽  
Author(s):  
R. Prisca Massengo-Tiassé ◽  
John E. Cronan
Keyword(s):  
Fatty Acid ◽  
Vibrio Cholerae ◽  
Mutant Strain ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
Gene Encoding ◽  
E Coli ◽  
New Class ◽  
Enoyl Acp Reductase

Enoyl-acyl carrier protein (ACP) reductase catalyzes the last step of the fatty acid elongation cycle. The paradigm enoyl-ACP reductase is the FabI protein of Escherichia coli that is the target of the antibacterial compound, triclosan. However, some Gram-positive bacteria are naturally resistant to triclosan due to the presence of the triclosan-resistant enoyl-ACP reductase isoforms, FabK and FabL. The genome of the Gram-negative bacterium, Vibrio cholerae lacks a gene encoding a homologue of any of the three known enoyl-ACP reductase isozymes suggesting that this organism encodes a novel fourth enoyl-ACP reductase isoform. We report that this is the case. The gene encoding the new isoform, called FabV, was isolated by complementation of a conditionally lethal E. coli fabI mutant strain and was shown to restore fatty acid synthesis to the mutant strain both in vivo and in vitro. Like FabI and FabL, FabV is a member of the short chain dehydrogenase reductase superfamily, although it is considerably larger (402 residues) than either FabI (262 residues) or FabL (250 residues). The FabV, FabI and FabL sequences can be aligned, but only poorly. Alignment requires many gaps and yields only 15% identical residues. Thus, FabV defines a new class of enoyl-ACP reductase. The native FabV protein has been purified to homogeneity and is active with both crotonyl-ACP and the model substrate, crotonyl-CoA. In contrast to FabI and FabL, FabV shows a very strong preference for NADH over NADPH. Expression of FabV in E. coli results in markedly increased resistance to triclosan and the purified enzyme is much more resistant to triclosan than is E. coli FabI.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

2018 ◽  
Vol 40 (4) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Recombinant Antibody ◽  
Blood Type ◽  
Soluble Form ◽  
Supernatant Fraction ◽  
Blood Group Antigens ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Single Chain Variable Fragments

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

Large-scale High-throughput Screening Revealed 5'-(carbonylamino)-2,3'- bithiophene-4'-carboxylate as Novel Template for Antibacterial Agents

2020 ◽  
Vol 17 (5) ◽  
pp. 716-724
Author(s):  
Yan A. Ivanenkov ◽  
Renat S. Yamidanov ◽  
Ilya A. Osterman ◽  
Petr V. Sergiev ◽  
Vladimir A. Aladinskiy ◽  
...  
Keyword(s):  
Antibacterial Activity ◽  
High Throughput ◽  
High Throughput Screening ◽  
Large Scale ◽  
Antibacterial Agents ◽  
Sos Response ◽  
Luciferase Assay ◽  
Translational Machinery ◽  
E Coli ◽  
New Class

Background: The key issue in the development of novel antimicrobials is a rapid expansion of new bacterial strains resistant to current antibiotics. Indeed, World Health Organization has reported that bacteria commonly causing infections in hospitals and in the community, e.g. E. Coli, K. pneumoniae and S. aureus, have high resistance vs the last generations of cephalosporins, carbapenems and fluoroquinolones. During the past decades, only few successful efforts to develop and launch new antibacterial medications have been performed. This study aims to identify new class of antibacterial agents using novel high-throughput screening technique. Methods: We have designed library containing 125K compounds not similar in structure (Tanimoto coeff.< 0.7) to that published previously as antibiotics. The HTS platform based on double reporter system pDualrep2 was used to distinguish between molecules able to block translational machinery or induce SOS-response in a model E. coli system. MICs for most active chemicals in LB and M9 medium were determined using broth microdilution assay. Results: In an attempt to discover novel classes of antibacterials, we performed HTS of a large-scale small molecule library using our unique screening platform. This approach permitted us to quickly and robustly evaluate a lot of compounds as well as to determine the mechanism of action in the case of compounds being either translational machinery inhibitors or DNA-damaging agents/replication blockers. HTS has resulted in several new structural classes of molecules exhibiting an attractive antibacterial activity. Herein, we report as promising antibacterials. Two most active compounds from this series showed MIC value of 1.2 (5) and 1.8 μg/mL (6) and good selectivity index. Compound 6 caused RFP induction and low SOS response. In vitro luciferase assay has revealed that it is able to slightly inhibit protein biosynthesis. Compound 5 was tested on several archival strains and exhibited slight activity against gram-negative bacteria and outstanding activity against S. aureus. The key structural requirements for antibacterial potency were also explored. We found, that the unsubstituted carboxylic group is crucial for antibacterial activity as well as the presence of bulky hydrophobic substituents at phenyl fragment. Conclusion: The obtained results provide a solid background for further characterization of the 5'- (carbonylamino)-2,3'-bithiophene-4'-carboxylate derivatives discussed herein as new class of antibacterials and their optimization campaign.

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