Molecular Cloning, Expression, Purification, and Functional Characterization of Dammarenediol Synthase fromPanax ginseng

The objective of this study is to clone and charecterize the expression of dammarenediol synthase gene and then to determine the relationship between the expression of dammarenediol synthase gene that is involved in the ginsenoside biosynthetic pathway and the ginsenoside content. A cDNA phage library was constructed from a five-year-old ginseng root. The cDNA library was screened for the dammarenediol synthase gene by using its specific primers. It was further cloned and expressed in pET-30a vector. The recombinant plasmid pET-30a-DS was expressed in RosettaE. coli. The recombinant DS protein was purified by affinity chromatography. The production of dammarenediol was detected by liquid chromatography-mass spectrometry (LC-MS). Results showed that dammarenediol synthase gene was cloned from the cDNA library and was expressed in RosettaE. coliand the SDS-PAGE analysis showed the presence of purified DS protein. LS-MS showed the activity of DS protein, as the protein content increases the dammarenediol increases. Our results indicate that the recombinant dammarenediol synthase protein could increase the production of dammarenediol and the expression of DS played a vital role in the biosynthesis of ginsenosides inP. ginseng.

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Functional Characterization of Porcine NK-Lysin: A Novel Immunomodulator That Regulates Intestinal Inflammatory Response

Molecules ◽

10.3390/molecules26144242 ◽

2021 ◽

Vol 26 (14) ◽

pp. 4242

Author(s):

Qian Lin ◽

Qingqing Fu ◽

Daiwen Chen ◽

Bing Yu ◽

Yuheng Luo ◽

...

Keyword(s):

Inflammatory Responses ◽

Functional Characterization ◽

Antibacterial Activities ◽

Bacterial Strains ◽

E Coli ◽

Gram Positive Bacteria ◽

Tnf Α ◽

Sds Page ◽

Junction Protein

Porcine NK-Lysine (PNKL) is a new antimicrobial peptide (AMP) identified in the small intestine. In this study, PNKL protein was obtained through heterologous expression in Escherichia coli and was estimated by SDS-PAGE at 33 kDa. The antibacterial activities of PNKL were determined using various bacterial strains and showed broad-spectrum antimicrobial activity against Gram-negative and Gram-positive bacteria. Furthermore, E. coli K88-challenged IPEC-J2 cells were used to determine PNKL influences on inflammatory responses. Hemolytic assays showed that PNKL had no detrimental impact on cell viability. Interestingly, PNKL elevated the viability of IPEC-J2 cells exposure to E. coli K88. PNKL significantly decreased the cell apoptosis rate, and improved the distribution and abundance of tight junction protein ZO-1 in IPEC-J2 cells upon E. coli K88-challenge. Importantly, PNKL not only down regulated the expressions of inflammatory cytokines such as the IL-6 and TNF-α, but also down regulated the expressions of NF-κB, Caspase3, and Caspase9 in the E. coli K88-challenged cells. These results suggest a novel function of natural killer (NK)-lysin, and the anti-bacterial and anti-inflammatory properties of PNKL may allow it a potential substitute for conventionally used antibiotics or drugs.

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Expression and functional characterization of a novel antimicrobial peptide: human beta-defensin118

10.21203/rs.3.rs-17411/v1 ◽

2020 ◽

Author(s):

Qian Lin ◽

KunHong Xie ◽

Daiwen Chen ◽

Bing Yu ◽

Xiangbing Mao ◽

...

Keyword(s):

Inflammatory Responses ◽

Functional Characterization ◽

Antimicrobial Activities ◽

Bacterial Strains ◽

E Coli ◽

Gram Positive Bacteria ◽

Sds Page ◽

Novel Host ◽

Late Apoptosis

Abstract Background: β-defensin 118 (DEFB118 ) is a novel host defence peptide (HDP) identified in human. To evaluate its potentials for future utilization, the DEFB118 gene was expressed in Escherichia coli ( E. coli ) and the recombinant protein was fully characterized. Methods: The DEFB118 protein was obtained by heterologous expression using E. coli Rosetta (DE3). Antibacterical activity of DEFB118 were determined by using various bacterial strains. IPEC-J cells challenged by E. coli K88 were used to determine its influences on inflammatory responses. Results: The E. coli transformants yielded more than 250 mg/mL D EFB118 protein after 4 h induction by 1.0 mM IPTG. The DEFB118 was estimated by SDS-PAGE to be 30 kDa, and MALDI-TOF analysis verified it is a human β-defensin 118. Importantly, the DEFB118 showed antimicrobial activities against both Gram-negative bacteria ( E. coli K88 and E. coli DH5α) and Gram-positive bacteria ( S. aureus and B. subtilis ), with a minimum inhibitory concentration (MIC) of 4 μg/mL. Hemolytic assays showed that DEFB118 had no detrimental impact on cell viability. Additionally, DEFB118 was found to elevate the viability of IPEC-J2 cells upon E. coli K88 challenge. Moreover, DEFB118 significantly decreased cell apoptosis in the late apoptosis phase and down-regulated the expression of inflammatory cytokines such as the IL-1β and TNF-a in the IPEC-J2 cells exposure to E. coli K88. Conclusions: These results suggested a novel function of the mammalian defensins, and the anti-bacterial and anti-inflammatory properties of DEFB118 may allow it a potential substitute for conventionally used antibiotics or drugs.

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Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽

10.1099/mic.0.28753-0 ◽

2006 ◽

Vol 152 (7) ◽

pp. 2129-2135 ◽

Cited By ~ 17

Author(s):

Taku Oshima ◽

Francis Biville

Keyword(s):

Functional Characterization ◽

Anaerobic Growth ◽

Functional Identification ◽

New Members ◽

E Coli ◽

Inner Membrane Protein ◽

Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

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Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli

JBIC Journal of Biological Inorganic Chemistry ◽

10.1007/s00775-021-01854-y ◽

2021 ◽

Author(s):

I. B. Trindade ◽

G. Hernandez ◽

E. Lebègue ◽

F. Barrière ◽

T. Cordeiro ◽

...

Keyword(s):

Functional Characterization ◽

Paramagnetic Nmr ◽

E Coli ◽

The Past ◽

Internal Cavities ◽

Structural Differences ◽

Bond Strengths ◽

Redox Bohr Effect ◽

Micromolar Range

AbstractIron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the “palm” domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe–2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins. Graphic abstract

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Sa1806 Molecular and Functional Characterization of Fimh in Crohn's Disease Associated E. Coli

Gastroenterology ◽

10.1016/s0016-5085(13)61117-8 ◽

2013 ◽

Vol 144 (5) ◽

pp. S-310

Author(s):

Brendan Chandler ◽

Belgin Dogan ◽

Ellen J. Scherl ◽

Kenneth W. Simpson

Keyword(s):

Crohn’S Disease ◽

Crohn's Disease ◽

Functional Characterization ◽

E Coli

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Expression, secretion and functional characterization of three laccases in E. coli

Synthetic and Systems Biotechnology ◽

10.1016/j.synbio.2021.12.002 ◽

2022 ◽

Vol 7 (1) ◽

pp. 474-480

Author(s):

Yating Mo ◽

Hou Ip Lao ◽

Sau Wa Au ◽

Ieng Chon Li ◽

Jeremy Hu ◽

...

Keyword(s):

Functional Characterization ◽

E Coli

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Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels

Physiological Genomics ◽

10.1152/physiolgenomics.00026.2006 ◽

2006 ◽

Vol 26 (1) ◽

pp. 1-7 ◽

Cited By ~ 14

Author(s):

Si Sun ◽

Jo Han Gan ◽

Jennifer J. Paynter ◽

Stephen J. Tucker

Keyword(s):

Functional Properties ◽

Functional Characterization ◽

Unicellular Eukaryote ◽

Channel Blockers ◽

E Coli ◽

Inwardly Rectifying Potassium Channels ◽

Genes Encoding ◽

Functionally Diverse ◽

Inwardly Rectifying

Our understanding of the mammalian inwardly rectifying family of K+ channels (Kir family) has recently been advanced by X-ray crystal structures of two homologous prokaryotic orthologs (KirBac1.1 and KirBac3.1). However, the functional properties of these KirBac channels are still poorly understood. To address this problem, we cloned and characterized genes encoding KirBac orthologs from a wide variety of different prokaryotes and a simple unicellular eukaryote. The functional properties of these KirBacs were then examined by growth complementation in a K+ uptake-deficient strain of Escherichia coli (TK2420). Whereas some KirBac genes exhibited robust growth complementation, others either did not complement or showed temperature-dependent complementation including KirBac1.1 and KirBac3.1. In some cases, KirBac expression was also toxic to the growth of E. coli. The KirBac family exhibited a range of sensitivity to the K+ channel blockers Ba2+ and Cs+ as well as differences in their ability to grow on very low-K+ media, thus demonstrating major differences in their permeation properties. These results reveal the existence of a functionally diverse superfamily of microbial KirBac genes and present an excellent resource for the structural and functional analysis of this class of K+ channels. Furthermore, the complementation assay used in this study provides a simple and robust method for the functional characterization of a range of prokaryotic K+ channels that are difficult to study by traditional methods.

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Novel biochemical and functional insights into nuclear Ca2+ transport through IP3Rs and RyRs in osteoblasts

AJP Renal Physiology ◽

10.1152/ajprenal.2000.278.5.f784 ◽

2000 ◽

Vol 278 (5) ◽

pp. F784-F791 ◽

Cited By ~ 21

Author(s):

Olugbenga A. Adebanjo ◽

Gopa Biswas ◽

Baljit S. Moonga ◽

Hindupur K. Anandatheerthavarada ◽

Li Sun ◽

...

Keyword(s):

Gene Expression ◽

Nuclear Envelope ◽

Nuclear Membrane ◽

Ryanodine Receptors ◽

Functional Characterization ◽

Inositol Trisphosphate Receptors ◽

Nuclear Membranes ◽

Sds Page ◽

Direct Regulation

We report the first biochemical and functional characterization of inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) in the nuclear membrane of bone-forming (MC3T3-E1) osteoblasts. Intact nuclei fluoresced intensely with anti-RyR (Ab34) and anti-IP3R (Ab40) antisera in a typically peripheral nuclear membrane pattern. Isolated nuclear membranes were next subjected to SDS-PAGE and blotted with isoform-specific anti-receptor antisera, notably Ab40, anti-RyR-1, anti-RyR-2 (Ab129), and anti-RyR-3 (Ab180). Only anti-RyR-1 and Ab40 showed bands corresponding, respectively, to full-length RyR-1 (∼500 kDa) and IP3R-1 (∼250 kDa). Band intensity was reduced by just ∼20% after brief tryptic proteolysis of intact nuclei; this confirmed that isolated nuclear membranes were mostly free of endoplasmic reticular contaminants. Finally, the nucleoplasmic Ca2+ concentration ([Ca2+]np) was measured in single nuclei by using fura-dextran. The nuclear envelope was initially loaded with Ca2+ via Ca2+-ATPase activation (1 mM ATP and ∼100 nM Ca2+). Adequate Ca2+ loading was next confirmed by imaging the nuclear envelope (and nucleoplasm). Exposure of Ca2+-loaded nuclei to IP3 or cADP ribose resulted in a rapid and sustained [Ca2+]np elevation. Taken together, the results provide complementary evidence for nucleoplasmic Ca2+ influx in osteoblasts through nuclear membrane-resident IP3Rs and RyRs. Our findings may conceivably explain the direct regulation of osteoblastic gene expression by hormones that use the IP3-Ca2+pathway.

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Functional characterization of the Haemophilus influenzae 4.5S RNA

Canadian Journal of Microbiology ◽

10.1139/w97-124 ◽

1998 ◽

Vol 44 (1) ◽

pp. 91-94

Author(s):

G Scott Jenkins ◽

Mark S Chandler ◽

Pamela S Fink

Keyword(s):

Haemophilus Influenzae ◽

Functional Characterization ◽

Coli Strain ◽

Northern Analysis ◽

Gene Encoding ◽

E Coli ◽

Functional Homolog ◽

Polymerase Chain ◽

4.5S Rna

The putative 4.5S RNA of Haemophilus influenzae was identified in the genome by computer analysis, amplified by the polymerase chain reaction, and cloned. We have determined that this putative 4.5S RNA will complement an Escherichia coli strain conditionally defective in 4.5S RNA production. The predicted secondary structures of the molecules were quite similar, but Northern analysis showed that the H. influenzae RNA was slightly larger than the E. coli RNA. The H. influenzae gene encoding this RNA is the functional homolog of the ffs gene in E. coli. Key words: ffs gene, complementation studies, small RNA, prokaryotic genetics.

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