• Title/Summary/Keyword: coexpression

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Domain Expression of ErmSF, MLS (macrolide-lincosamide-streptogramin B) Antibiotic Resistance Factor Protein (MLS (macrolide-lincosamide-streptogramin B) 항생제 내성인자 단백질인 ErmSF의 domain발현)

  • 진형종
    • Korean Journal of Microbiology
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    • v.37 no.4
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    • pp.245-252
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    • 2001
  • Erm proteins, MLS (macrolide-lincosamide-streptogramin B) resistance factor proteins, show high degree of amino acid sequence homology and comprise of a group of structurally homologous N-methyltransferases. On the basis of the recently determined structures of ErmC` and ErmAM, ErmSF was divided into two domains, N-terminal end catalytic domain and C-terminal end substrate binding domain and attempted to overexpress catalytic domain in E. coli using various pET expression systems. Three DNA fragments were used to express the catalytic domain: DNA fragment 1 encoding Met 1 through Glu 186, DNA fragment 2 encoding Arg 60 to Glu 186 and DNA fragment 3 encoding Arg 60 through Arg 240. Among the pET expression vectors used, pET 19b successfully expressed the DNA fragment 3 and pET23b succeeded in expression of DNA fragment 1 and 2. But the overexpressed catalytic domains existed as inclusion body, a insoluble aggregate. To assist the soluble expression of ErmSF catalytic domains, Coexpression of chaperone GroESL or Thioredoxin and lowering the incubation temperature to $22^{\circ}C$ were attempted, as did in the soluble expression of the whole ErmSF protein. Both strategies did not seem to be helpful. Solubilization with guanidine-HCl and renaturation with gradual removal of denaturant and partial digestion of overexpressed whole ErmSF protein (expressed to the level of 126 mg/ι culture as a soluble protein) with proteinase K, nonspecific proteinase are under way.

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Cloning of tlrD, 23S rRNA Monomethyltransferase Gene, Overexpression in Eschepichia coli and Its Activity (235 rRNA Monomethyltransferase인 tlrD의 클로닝, 이의 대장균에서 대량생산과 활성 검색)

  • Jin, Hyung-Jong
    • Korean Journal of Microbiology
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    • v.43 no.3
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    • pp.166-172
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    • 2007
  • ERM proteins transfer the methyl group to $A_{2058}$ in 23S rRNA, which reduces the affinity of MLS (macrolide-lincosamide-streptogramin B) antibiotics to 23S rRNA, thereby confer the antibiotic resistance on micro-organisms ranging from antibiotic producers to pathogens and are classified into monomethyltransferase and dimethyltransferase. To investigate the differences between mono- and dimethyltransferase, tirD, a representative monomethylase gene was cloned in Escherichia coli from Streptomyces fradiae which contains ermSF, dimethylase gene as well to overexpress the TlrD for the first time. T7 promoter driven expression system successfully overexpress tlrD as a insoluble aggregate at $37^{\circ}C$ accumulating to around 55% of the total cell protein but unlike ErmSF, culturing at temperature as low as $18^{\circ}C$ did not make insoluble aggregate of protein into soluble protein. Coexpression of Thioredoxin and GroESL, chaperone was not helpful in turning into soluble protein either as in case of ErmSF. These results might suggest that differences between mono- and dimethylase could be investigated on the basis of the characteristics of protein structure. However, a very small amount of soluble protein which could not be detected by SDS-PAGE conferred antibiotic resistance on E. coli as in ErmSF which was expected from the activity exerted by monmethylase in a cell.

Implications of Streptomyces coelicolor RraAS1 as an activator of ribonuclease activity of Escherichia coli RNase E (Streptomyces coelicolor RraAS1의 Eschechia coli RNase E의 RNA 분해작용에 대한 활성제로서 기능 암시)

  • Heo, Jihune;Seo, Sojin;Lee, Boeun;Yeom, Ji-Hyun;Lee, Kangseok
    • Korean Journal of Microbiology
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    • v.52 no.3
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    • pp.243-248
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    • 2016
  • RNase E (Rne) is an essential enzyme involved in the processing and degradation of a large portion of RNAs in Escherichia coli. The enzymatic activity of RNase E is controlled by regulators of ribonuclease activity, namely, RraA and RraB. Gram-positive bacterium Streptomyces coelicolor also contains homologs of Rne and RraA, designated as RNase ES (Rns), RraAS1, and RraAS2. In the present study, we investigated the effect of S. coelicolor RraAS1 on the ribonucleolytic activity of RNase E in E. coli. Coexpression of RraAS1 with Rne resulted in the decreased levels of rpsO, ftsZ, and rnhB mRNAs, which are RNase E substrates, and augmented the toxic effect of Rne overexpression on cell growth. These in vivo effects appeared to be induced by the binding of RraAS1 to Rne, as indicated by the results of co-immunoprecipitation analysis. These results suggested that RraAS1 induces ribonucleolytic activity of RNase E in E. coli.

Cloning of a Novel vpr Gene Encoding a Minor Fibrinolytic Enzyme from Bacillus subtilis SJ4 and the Properties of Vpr

  • Yao, Zhuang;Meng, Yu;Le, Huong Giang;Lee, Se Jin;Jeon, Hye Sung;Yoo, Ji Yeon;Kim, Hyun-Jin;Kim, Jeong Hwan
    • Journal of Microbiology and Biotechnology
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    • v.30 no.11
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    • pp.1720-1728
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    • 2020
  • We have previously characterized AprESJ4, the major fibrinolytic enzyme from Bacillus subtilis SJ4 (Yao et al., 2019). During that study, we observed a 68 kDa protein with fibrinolytic activity. In this study, we cloned the gene (vprSJ4) encoding the 68 kDa protein, a mature Vpr and minor protease secreted by Bacillus species. vprSJ4 encodes a preproenzyme consisting of 810 amino acids (aa) including signal sequence (28 aa) and prosequence (132 aa). The mature enzyme (650 aa) has a predicted molecular weight of 68,467.35. Unlike Vprs from other B. subtilis strains, VprSJ4 has 4 additional amino acids (DEFA) at the C-terminus. vprSJ4 was overexpressed in Escherichia coli. PreproVprSJ4 was localized in inclusion bodies, and subjected to in vitro renaturation and purification by an affinity column. SDS-PAGE and western blot showed that autoprocessing of preproVprSJ4 occurred and 68 kDa and smaller proteins were produced. The optimum pH and temperature of the recombinant VprSJ4 were pH 7.0 and 40℃, respectively. Kinetic parameters of recombinant VprSJ4 were measured by using an artificial substrate, N-succinyl-ala-ala-pro-phe-p-nitroanilide. Coexpression of vprSJ4 and aprESJ4 using pHY300PLK increased the fibrinolytic activity a further 117% when compared with aprESJ4 single expression using the same vector in B. subtilis WB600.

Time-based Expression Networks of Genes Related to Cold Stress in Brassica rapa ssp. pekinensis (배추의 저온 스트레스 처리 시간대별 발현 유전자 네트워크 분석)

  • Lee, Gi-Ho;Yu, Jae-Gyeong;Park, Young-Doo
    • Horticultural Science & Technology
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    • v.33 no.1
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    • pp.114-123
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    • 2015
  • Plants can respond and adapt to cold stress through regulation of gene expression in various biochemical and physiological processes. Cold stress triggers decreased rates of metabolism, modification of cell walls, and loss of membrane function. Hence, this study was conducted to construct coexpression networks for time-based expression pattern analysis of genes related to cold stress in Chinese cabbage (Brassica rapa ssp. pekinensis). B. rapa cold stress networks were constructed with 2,030 nodes, 20,235 edges, and 34 connected components. The analysis suggests that similar genes responding to cold stress may also regulate development of Chinese cabbage. Using this network model, it is surmised that cold tolerance is strongly related to activation of chitinase antifreeze proteins by WRKY transcription factors and salicylic acid signaling, and to regulation of stomatal movement and starch metabolic processes for systemic acquired resistance in Chinese cabbage. Moreover, within 48 h, cold stress triggered transition from vegetative to reproductive phase and meristematic phase transition. In this study, we demonstrated that this network model could be used to precisely predict the functions of cold resistance genes in Chinese cabbage.

Genome-wide identification and analysis of long noncoding RNAs in longissimus muscle tissue from Kazakh cattle and Xinjiang brown cattle

  • Yan, Xiang-Min;Zhang, Zhe;Liu, Jian-Bo;Li, Na;Yang, Guang-Wei;Luo, Dan;Zhang, Yang;Yuan, Bao;Jiang, Hao;Zhang, Jia-Bao
    • Animal Bioscience
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    • v.34 no.11
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    • pp.1739-1748
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    • 2021
  • Objective: In recent years, long noncoding RNAs (lncRNAs) have been identified in many species, and some of them have been shown to play important roles in muscle development and myogenesis. However, the differences in lncRNAs between Kazakh cattle and Xinjiang brown cattle remain undefined; therefore, we aimed to confirm whether lncRNAs are differentially expressed in the longissimus dorsi between these two types of cattle and whether differentially expressed lncRNAs regulate muscle differentiation. Methods: We used RNA-seq technology to identify lncRNAs in longissimus muscles from these cattle. The expression of lncRNAs were analyzed using StringTie (1.3.1) in terms of the fragments per kilobase of transcript per million mapped reads values of the encoding genes. The differential expression of the transcripts in the two samples were analyzed using the DESeq R software package. The resulting false discovery rate was controlled by the Benjamini and Hochberg's approach. KOBAS software was utilized to measure the expression of different genes in Kyoto encyclopedia of genes and genomes pathways. We randomly selected eight lncRNA genes and validated them by quantitative reverse transcription polymerase chain reaction (RT-qPCR). Results: We found that 182 lncRNA transcripts, including 102 upregulated and 80 downregulated transcripts, were differentially expressed between Kazakh cattle and Xinjiang brown cattle. The results of RT-qPCR were consistent with the sequencing results. Enrichment analysis and functional annotation of the target genes revealed that the differentially expressed lncRNAs were associated with the mitogen-activated protein kinase, Ras, and phosphatidylinositol 3-kinase (PI3k)/Akt signaling pathways. We also constructed a lncRNA/mRNA coexpression network for the PI3k/Akt signaling pathway. Conclusion: Our study provides insights into cattle muscle-associated lncRNAs and will contribute to a more thorough understanding of the molecular mechanism underlying muscle growth and development in cattle.

Production and characterization of lentivirus vector-based SARS-CoV-2 pseudoviruses with dual reporters: Evaluation of anti-SARS-CoV-2 viral effect of Korean Red Ginseng

  • Jeonghui Moon;Younghun Jung;Seokoh Moon;Jaehyeon Hwang;Soomin Kim;Mi Soo Kim;Jeong Hyeon Yoon;Kyeongwon Kim;Youngseo Park;Jae Youl Cho;Dae-Hyuk Kweon
    • Journal of Ginseng Research
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    • v.47 no.1
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    • pp.123-132
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    • 2023
  • Background: Pseudotyped virus systems that incorporate viral proteins have been widely employed for the rapid determination of the effectiveness and neutralizing activity of drug and vaccine candidates in biosafety level 2 facilities. We report an efficient method for producing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pseudovirus with dual luciferase and fluorescent protein reporters. Moreover, using the established method, we also aimed to investigate whether Korean Red Ginseng (KRG), a valuable Korean herbal medicine, can attenuate infectivity of the pseudotyped virus. Methods: A pseudovirus of SARS-CoV-2 (SARS-2pv) was constructed and efficiently produced using lentivirus vector systems available in the public domain by the introduction of critical mutations in the cytoplasmic tail of the spike protein. KRG extract was dose-dependently treated to Calu-3 cells during SARS2-pv treatment to evaluate the protective activity against SARS-CoV-2. Results: The use of Calu-3 cells or the expression of angiotensin-converting enzyme 2 (ACE2) in HEK293T cells enabled SARS-2pv infection of host cells. Coexpression of transmembrane protease serine subtype 2 (TMPRSS2), which is the activator of spike protein, with ACE2 dramatically elevated luciferase activity, confirming the importance of the TMPRSS2-mediated pathway during SARS-CoV-2 entry. Our pseudovirus assay also revealed that KRG elicited resistance to SARS-CoV-2 infection in lung cells, suggesting its beneficial health effect. Conclusion: The method demonstrated the production of SARS-2pv for the analysis of vaccine or drug candidates. When KRG was assessed by the method, it protected host cells from coronavirus infection. Further studies will be followed for demonstrating this potential benefit.

Coexpression of $P2X_3$ with TRPV1 in the Rat Trigeminal Sensory Nuclei (흰쥐 삼차신경감각핵에서 $P2X_3$와 TRPV1의 공존에 관한 연구)

  • Moon, Yong-Suk;Ryoo, Chang-Hyun;Cho, Yi-Sul;Kim, Hong-Tae;Park, Mae-Ja;Paik, Sang-Kyoo;Moon, Che-Il;Kim, Yun-Sook;Bae, Yong-Chul
    • Applied Microscopy
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    • v.38 no.3
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    • pp.151-157
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    • 2008
  • Trigeminal primary afferents expressing $P2X_3$ or transient receptor potential vanilloid 1 (TRPV1) are involved in the transmission of nociceptive information. In order to characterize $P2X_3$- and TRPV1-immunopositive neurons in the trigeminal ganglion (TG) and trigeminal caudal nucleus (Vc), we performed immunofluorescence experiments using anti-$P2X_3$ and anti-TRPV1 antisera and a morphometric analysis. 77.4% (1,401/1.801) of all the $P2X_3$-postive neurons coexpressed TRPV1 and 51.9% (1,401/2,698) of all the THFV1-immunopositive neurons also costained for $P2X_3$ in the TG. Immunoreactivity for both $P2X_3$ and TRPV1 were present in medium-sized neurons but not in small- and large-sized neurons. $P2X_3$ and/or TRPV1-immunopositive fibers were observed in the primary afferents and their associated axons in the Vc. These fibers and terminals were distributed in the superficial lamina of Vc: $P2X_3$-immunopositive fibers and terminals were distributed in the lamina I and II, expecially in the inner part of lamina II (lamina IIi), whereas TRPV1-immunopositive ones were densely detected in the lamina I and outer part of lamina II (lamina IIo). Immunopositive fibers and terminals for both $P2X_3$ and TRPV1 were observed on the border between lamina IIi and IIo. These results suggest that terminals coexpressing $P2X_3$ and TRPV1 are involved in specific roles in the transmission and processing of orofacial nociceptive information.

Characterization of Trigeminal Ganglion Neurons Expressing Transient Receptor Potential Ankyrin 1 (TRPA1) in the Rat (흰쥐의 삼차신경절에서 Transient receptor potential ankyrin 1 (TRPA1)의 발현 특성에 관한 연구)

  • Paik, Sang-Kyoo;Na, Yeon-Kyung;Kim, Yun-Sook
    • Applied Microscopy
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    • v.42 no.1
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    • pp.27-33
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    • 2012
  • Transient receptor potential ankyrin 1 (TRPA1), responding to noxious cold (${\leq}17^{\circ}C$) and pungent compounds, is implicated in nociception, but little is known about the coexpression of TRPA1 and other channels or receptors involved in the nociception in craniofacial regions. To address this issue, we characterized the TRPA1-immunopositive (+) neurons in the rat trigeminal ganglion (TG) and investigated their colocalization with other proteins known to be expressed in nociceptive neurons, such as transient receptor potential vanilloid (TRPV1) and $P2X_3$ receptor, using light microscopic immunofluorescence labeling method with TRPA1 and TRPV1 or $P2X_3$ antisera. The majority of TRPA1+ neurons costained for TRPV1 (TRPV1+/TRPA1+; 58.8%, 328/558) and 41.2% only expressed TRPA1 but not TRPV1. The TRPV1+/TRPA1+ neurons were small and medium sized. In addition, we investigated the colocalization of TRPA1 with $P2X_3$, a nonselective cation channel activated by ATP that may be released in the extracellular space as a result of tissue damage and inflammation. Among all TRPA1+ TG neurons, 26.1% (310/1186) costained for $P2X_3$, whereas 73.9% (876/1186) of TRPA1+ neurons did not coexpress $P2X_3$. $P2X_3$+/TRPA1+ neurons were predominantly small and medium sized. These results suggest that TRPA1+ neurons coexpressing TRPV1 or $P2X_3$ are involved in specific roles in the transmission and processing of orofacial nociceptive information by noxious cold, heat, and inflammation.