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 the recombinant single chain variable fragments recognizing blood antigen fused with thioredoxin in Escherichia coli

2019 ◽  
Vol 41 (1) ◽  
Author(s):  
Dang Thi Ngoc Ha ◽  
Le Thi Thu Hong ◽  
Truong Nam Hai
Keyword(s):  
Expression Vector ◽  
Recombinant Antibody ◽  
Soluble Form ◽  
Single Chain Variable Fragment ◽  
Single Chain ◽  
E Coli ◽  
Recombinant Scfv ◽  
Insoluble Form ◽  
Single Chain Variable Fragments

The technology of recombinant single chain variable fragments (scFvs) expression has been used in research, diagnosis and treatment of diseases. In the previous study, we studied the expression of a recombinant single chain variable fragment recognizing blood A antigen (antiA-scFv) in E. coli. However, the protein was insoluble form resulting in difficulty for purification, refolding and activity assesment. Here, we present the study on fused expression of the recombinant scFv -specific blood A antigen with thioredoxin (Trx) in the expression vector pET32a (+). The results showed that the Trx/antiA-scFv fusion protein was expressed with molecular weight of 49 kDa in a soluble form reaching 40% of the total recombinant protein. This result facilitates the optimal condition of soluble protein expression, purification and bioactivity determination of the antiA-scFv recombinant antibody. 

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 SUBKLONING DAN ISOLASI GEN PENYANDI MIKRONEMA 3 (MIC-3) Toxoplasma gondii ISOLAT LOKAL

Molekul ◽  
2011 ◽  
Vol 6 (1) ◽  
pp. 10
Author(s):  
Diana Indrasanti ◽  
Aris Haryanto ◽  
Wayan T. Artama
Keyword(s):  
Toxoplasma Gondii ◽  
Expression Vector ◽  
Recombinant Dna ◽  
Cell Infection ◽  
Recombinant Dna Technology ◽  
Specific Primers ◽  
Gene Encoding ◽  
E Coli ◽  
Local Isolate ◽  
Microneme Protein

Microneme protein (MIC) is one of proteins that belongs to excretory-secretory antigens (ESAs) of Toxoplasma gondii. Microneme 3 protein (MIC-3) is the protein that plays an important role in the invasion proccess during cell infection as a mediator attachment parasite to the host cell. The aim of this research is to clone mic3 (gene encoding for MIC-3) of T. gondii from local isolate using recombinant DNA technology by cloning mic3 in an expression vector. Deoxyribonucleic acid (DNA) from T. gondii tachyzoites was amplified by PuRe Taq RTG-PCR Beads using mic3 specific primers. Amplified DNA was double digested using EcoRV and HindIII restriction endonucleases and then purified using EZ-10 spin coloumn purification kit. The mic3 DNA was ligated into pET-32a(+) expression vector and transformated into Escherichia coli BL21. The results showed that recombinant mic3gene 4.2 kDa has been successfully performed by cloning gene encoding for MIC-3 protein of T. gondii local isolate into pET-32a(+) and transformed to E. coli BL21.

The Molecular basis of N-acetylmuramoyl-L-alanine amidase (Rv3915) and Protein Kinase B (PknB) essentiality in Mycobacterium tuberculosis

2019 ◽  
Author(s):  
Ifunanya N Ezechukwu ◽  
Kenechukwu Onyekwelu ◽  
Loreta Nwokolo ◽  
Obolbek Turapov
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Protein Kinase ◽  
Soluble Fraction ◽  
Protein Kinase B ◽  
Sodium Dodecyl ◽  
Coli Strain ◽  
Gene Encoding ◽  
E Coli ◽  
Sds Page

Abstract Background: Protein kinase B (PknB) is critical for the survival of Mycobacterium tuberculosis (M. tuberculosis) in vitro and in hosts. It phosphorylates various enzymes involved in biosynthesis of cell wall and a particular autolysin classified as CwIM or Rv3915 has been recently identified as a PknB substrate. However, in-depth knowledge of this protein is still unknown. The aims of this study were to purify and investigate the activity of Rv3915, as well as monitor the phosphorylation of Rv3915 and the influence of phosphorylation on the activity of this protein. Results: Using the C41 E. coli strain containing a plasmid, with a gene encoding the protein, Rv3915 was either expressed alone or co-expressed with PknB. Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) was used to observe the amount of Rv3915 purified, anti-poly His and anti-phosphothreonine western blot techniques were used to confirm the presence and phosphorylation of Rv3915 and zymogram assays were run to examine its activity. The results showed that Rv3195 was successfully expressed and was deemed soluble when observed in soluble fraction of E. coli lysate. It was confirmed that Rv3915 is produced as a ~45kDa protein, which does not possess any muralytic activity. However a shorter version of the protein (~25kDA) was active in zymogram, suggesting that Rv3915 is activated by cleavage. Conclusion: Rv3915 was phosphorylated by PknB and phosphorylation apparently controls stability of the protein.

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.

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. 

scholarly journals Soluble Expression and Efficient Purification of Recombinant Class I Hydrophobin DewA

2021 ◽  
Vol 22 (15) ◽  
pp. 7843
Author(s):  
Sang-Oh Ahn ◽  
Ho-Dong Lim ◽  
Sung-Hwan You ◽  
Dae-Eun Cheong ◽  
Geun-Joong Kim
Keyword(s):  
Tertiary Structure ◽  
Signal Sequence ◽  
Soluble Expression ◽  
Class I ◽  
Soluble Form ◽  
Two Phase ◽  
E Coli ◽  
Small Proteins ◽  
Industrial Use ◽  
Shed Light

Hydrophobins are small proteins (<20 kDa) with an amphipathic tertiary structure that are secreted by various filamentous fungi. Their amphipathic properties provide surfactant-like activity, leading to the formation of robust amphipathic layers at hydrophilic–hydrophobic interfaces, which make them useful for a wide variety of industrial fields spanning protein immobilization to surface functionalization. However, the industrial use of recombinant hydrophobins has been hampered due to low yield from inclusion bodies owing to the complicated process, including an auxiliary refolding step. Herein, we report the soluble expression of a recombinant class I hydrophobin DewA originating from Aspergillus nidulans, and its efficient purification from recombinant Escherichia coli. Soluble expression of the recombinant hydrophobin DewA was achieved by a tagging strategy using a systematically designed expression tag (ramp tag) that was fused to the N-terminus of DewA lacking the innate signal sequence. Highly expressed recombinant hydrophobin DewA in a soluble form was efficiently purified by a modified aqueous two-phase separation technique using isopropyl alcohol. Our approach for expression and purification of the recombinant hydrophobin DewA in E. coli shed light on the industrial production of hydrophobins from prokaryotic hosts.

scholarly journals Novel Bacterial Production of Two Different Bioactive Forms of Human Stem-Cell Factor

2021 ◽  
Vol 22 (12) ◽  
pp. 6361
Author(s):  
Eunyoung Lee ◽  
Michelle Novais de Paula ◽  
Sangki Baek ◽  
Huynh Kim Khanh Ta ◽  
Minh Tan Nguyen ◽  
...  
Keyword(s):  
Stem Cell ◽  
Ion Exchange ◽  
Stem Cell Factor ◽  
Transmembrane Domain ◽  
Coli Strain ◽  
Exchange Chromatography ◽  
Soluble Form ◽  
Human Stem Cell ◽  
E Coli ◽  
Human Stem Cell Factor

Human stem-cell factor (hSCF) stimulates the survival, proliferation, and differentiation of hematopoietic cells by binding to the c-Kit receptor. Various applications of hSCF require the efficient and reliable production of hSCF. hSCF exists in three forms: as two membrane-spanning proteins hSCF248 and hSCF229 and truncated soluble N-terminal protein hSCF164. hSCF164 is known to be insoluble when expressed in Escherichia coli cytoplasm, requiring a complex refolding procedure. The activity of hSCF248 has never been studied. Here, we investigated novel production methods for recombinant hSCF164 and hSCF248 without the refolding process. To increase the solubility of hSCF164, maltose-binding protein (MBP) and protein disulfide isomerase b’a’ domain (PDIb’a’) tags were attached to the N-terminus of hSCF164. These fusion proteins were overexpressed in soluble form in the Origami 2(DE3) E. coli strain. These solubilization effects were enhanced at a low temperature. His-hSCF248, the poly-His tagged form of hSCF248, was expressed in a highly soluble form without a solubilization tag protein, which was unexpected because His-hSCF248 contains a transmembrane domain. hSCF164 was purified using affinity and ion-exchange chromatography, and His-hSCF248 was purified by ion-exchange and gel filtration chromatography. The purified proteins stimulated the proliferation of TF-1 cells. Interestingly, the EC50 value of His-hSCF248 was 1 pg/mL, 100-fold lower than 9 ng/mL hSCF164. Additionally, His-hSCF248 decreased the doubling time, increased the proportion of S and G2/M stages in the cell cycle, and increased the c-Myc expression at a 1000-fold lower concentration than hSCF164. In conclusion, His-hSCF248 was expressed in a soluble form in E. coli and had stronger activity than hSCF164. The molecular chaperone, MBP, enabled the soluble overexpression of hSCF164.

scholarly journals Cloning of Salmonella enterica Serovar Enteritidis Fimbrial Protein SefA as a Surface Protein in Escherichia coli Confers the Ability To Attach to Eukaryotic Cell Lines

2009 ◽  
Vol 75 (20) ◽  
pp. 6622-6625 ◽  
Author(s):  
Douglas L. Rank ◽  
Mahdi A. Saeed ◽  
Peter M. Muriana
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Expression Vector ◽  
Surface Protein ◽  
Surface Expression ◽  
Eukaryotic Cell ◽  
Salmonella Enterica Serovar Enteritidis ◽  
Western Blot Assay ◽  
E Coli ◽  
Fimbrial Protein

ABSTRACT The gene for the Salmonella enterica serovar Enteritidis fimbrial protein SefA was cloned into an Escherichia coli surface expression vector and confirmed by Western blot assay. E. coli clones expressing SefA attached to avian ovary granulosa cells and HEp-2 cells, providing evidence for the involvement of SefA in the ability of Salmonella to attach to eukaryotic cells.

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