• Title/Summary/Keyword: Recombinant

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Plasmid Stability and Cloned-Gene Expression in Continuous Culture of Recombinant Escherichia Coli Under Derepressed Condition

  • Nam, Soo-Wan;Kim, Byung-Kwan;Kim, Jung-Hoe
    • Journal of Microbiology and Biotechnology
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    • v.4 no.1
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    • pp.1-6
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    • 1994
  • Continuous culture was carried out with a recombinant Escherichia coli W3110/pCR185, which encodes trp-operon enzymes when the temperature is shifted from $37^{circ}C\;t;42^{\circ}C$. Under derepressed condition of $42^{\circ}C$. plasmlid stability and gene expression were analysed as function of the dilution rate. The stability of plasmid increased with the dilution rate, but maximal levels of gene expression (tryptophan concentration) and plasmid DNA content were obtained at the lowest dilution rate, $0.075\;hr^{-1}$. The plasmid instability, observed at low dilution rates, could be explained by the unbalanced biosynthetic state of the recombinant cell harboring a high copy number of plasmid.

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Insect Cell Culture for Recombinant $\beta$-galactosidase Production Using a Spin-filter Bioreactor

  • Chung, In-Sik;Kim, Hak-Ryul;Lee, Ki-Woong;Kim, Tae-Yong;Oh, Jai-Hyn;Yang, Jai-Myung
    • Journal of Microbiology and Biotechnology
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    • v.4 no.3
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    • pp.200-203
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    • 1994
  • Spodoptera frugiperda IPLB-SF-21-AE cells were cultivated in a spin-filter bioreactor with continuous perfusion for the recombinant $\beta$-galactosidase production. At the perfusion rate of 0.06 $hr^{-1}$, the maximum cell density of insect cells in this bioreactor system reached 3.5$\times$$l0^6$ viable cells/ml using the Grace media containing 5% FBS and 0.3% Pluronic F-68. The recombinant $\beta$-galactosidase production of 8, 100 units per reactor volume was also achieved at this perfusion rate.

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Comparison of enzymatic activities between the recombinant CHT1 proteins from the hard tick Haemaphysalis longicornis expressed in E. coli and baculovirus-mediated Sf 9 cells (E. coli와 baculovirus-mediated Sf 9 세포에서 발현된 진드기 H. longicornis의 CHT1 단백의 효소활성 비교)

  • You, Myung-Jo;Fujisaki, Kozo
    • Korean Journal of Veterinary Research
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    • v.43 no.1
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    • pp.139-144
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    • 2003
  • A chitinase cDNA named CHT1 was cloned from the hard tick, Haemaphysalis longicornis, and the enzymatic properties of its recombinant proteins were characterized. The CHT1 cDNA encodes 930 amino-acid (aa) residues including a 22 aa putative signal peptide, with the calculated molecular mass of the putative mature protein 104 kDa. The E coli-expressed rCHT1 exhibited weak chitinolytic activity against $4MU-(GlcNAc)_3$. The rCHT1 protein with higher activity was obtained using recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV), which expresses rCHT1 under polyhedrin promoter. These findings suggest that the rCHT1 expressed in baculovirus-mediated Sf 9 cells has a high activity than E coli-expressed rCHT1.

A Case of Pregnancy Using Recombinant Follicle Stimulating Hormone and Gonadotropin Releasing Hormone Antagonist (유전자 재조합 난포자극호르몬과 성선자극호르몬 분비호르몬 길항제를 이용한 임신 1례)

  • Nam, Yoon-Sung;Kim, Nam-Keun;Kim, Eun-Kyung;Chung, Hyung-Min;Cha, Kwang-Yul
    • Clinical and Experimental Reproductive Medicine
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    • v.28 no.1
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    • pp.73-77
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    • 2001
  • Objective: To report the pregnancy which was made by in vitro fertilization using recombinant follicle stimulating hormone and gonadotropin releasing hormone antagonist. Material and Method: Case report. Results: Six oocytes were retrieved and all were fertilized by intracytoplasmic sperm injection. Six embryos were transferred and the pregnancy was confirmed. Conclusion: It is envisaged that the availability of recombinant gonadotropins and gonadotropin releasing hormone antagonists will ultimately lead to shorter, cheaper and safer treatments, using reduced dosages.

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Acute Toxicity of Recombinant Human Epidermal Growth factor, DWP-401 in Mice (Recombinant Human Epidermal Growth Factor, DWP-401에 대한 마우스에서의 급성독성)

  • 김효정;서경원;오미현;선우유신;유영효;문병우
    • Biomolecules & Therapeutics
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    • v.2 no.1
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    • pp.77-81
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    • 1994
  • The acute toxicity of recombinant human epidermal growth factor, DWP-401 was evaluated in ICR mice of both sexes. Six groups of mice were administered orally or subcutaneously with 0, 0.125, 0.25, 0.5, 1 and 2 mg/kg of DWP-401. Abnormal clinical signs related to the compound were not observed, and no deaths occurred. Gross findings of necropsy revealed no evidence of specific toxicity related to DWP-401. LD$_{50}$ values for both male and female mice were evaluated to be over 2 mg/kg, which is approximately 2, 000 fold of presumed clinical dose.e.

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Acute Toxicity of Recombinant Human Epidermal Growth Factor, DWP-401 in Rats (Recombinant Human Epidermal Growth Factor, DWP-401의 랫드에서의 급성 독성)

  • 심점순;오미현;서경원;선우유신;이경민;김효정
    • Journal of Food Hygiene and Safety
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    • v.9 no.1
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    • pp.31-36
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    • 1994
  • The acute toxicity of recombinant human epidermal growth factor, DWP-401 was evaluated in SD rats. Male and female rats aging 6 weeks were administered orally or subcutaneously with 0, 0.125, 0.25, 0.5, 1 and 2 mg/kg of DWP-401. No deaths and no toxic symptoms related to the DWP-401 were observed. The body weights of treated animals were not significantly different from the controls. The results of necropsy revealed no abnormal gross findings of the organs in treated animals. LD50 values of DWP-401 for male and female rats were estimated to be over 2 mg/kg, which is approximately 2, 000 times of expected clinical dose.

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Simple Purification of Escherichia coli-Derived Recombinant Human Interleukin-2 Expressed with N-terminus Fusion of Glucagon

  • Won Hye-Soon;Lee Jeewon;Kim In-Ho;Park Young-Hoon
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.5 no.1
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    • pp.13-16
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    • 2000
  • Simple procedures have been devised for purifying recombinant human interleukin-2 (hIL-2), which was expressed in Escberichia coli using sequences of glucagon molecules and enterokinase cleavage site as an N-terminus fusion partner. The insoluble aggregates of recombinant fusion protein produced in E. coli cytoplasm were easily dissolved by simple alkaline pH shift $(8\rightarrow12\rightarrow8)$. Following enterokinase cleavage, the recombinant hIL-2 was finally purified by one-step reversed-phase HPLC with high purity. The ease and high efficiency of this simple purification process seem to mainly result from the role of used glucagon fusion partner, which could be applied to the production of other therapeutically important proteins.

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Replication and encapsidation of recombinant Turnip yellow mosaic virus RNA

  • Shin, Hyun-Il;Kim, In-Cheol;Cho, Tae-Ju
    • BMB Reports
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    • v.41 no.10
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    • pp.739-744
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    • 2008
  • Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus that infects mainly Cruciferae plants. In this study, the TYMV genome was modified by inserting an extra subgenomic RNA promoter and a multiple cloning site. This modified TYMV was introduced into Nicotiana benthamiana using a Agrobacterium-mediated T-DNA transfer system (agroinfiltration). When a gene encoding $\beta$-glucuronidase or green fluorescent protein was expressed using this modified TYMV as a vector, replication of the recombinant viruses, especially the virus containing $\beta$-glucuronidase gene, was severely inhibited. The suppression of replication was reduced by co-expression of viral silencing suppressor genes, such as tombusviral p19, closteroviral p21 or potyviral HC-Pro. As expected, two subgenomic RNAs were produced from the recombinant TYMV, where the larger one contained the foreign gene. An RNase protection assay revealed that the recombinant subgenomic RNA was encapsidated as efficiently as the genuine subgenomic RNA.

Separation and Purification of Useful Proteins Using Hydrogel Ultratiltration

  • Park, Chang-Ho;Son, Chang-Kyu;Park, Jong-Hwa;Chung, In-Sik
    • Biotechnology and Bioprocess Engineering:BBE
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    • v.2 no.2
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    • pp.113-116
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    • 1997
  • The hydrogel process is a different form of ultrafiltration and has been used to separate biological molecules. In this study, the gel pore size was predicted by pulse NMR technique and neural network using a database obtained from gel filtration chromatography and diffusion experiment. Recombinant alkaline phosphatase expressed in insect cells was concentratred 1.5 times by hydrogel ultrafiltration by swelling at 2$0^{\circ}C$ and collapsing at 35$^{\circ}C$ at 53-65% separation efficiency and 78-83% enzyme recovery. Wild and recombinant Autographa californica unclear polyhedrosis viruses (AcNPV) were also concentrated 1.4 and 1.6 times of the feed solution at 48.5 and 60.0% separation efficiency, respectively Hydrogel ultrafiltration appears to be an attractive alternative for the concentration of AcNPV and recombinant proteins from insect cells.

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Immunogenicity of Recombinant Outer Membrane Protein H from Pasteurella multocida (재조합 파스튜렐라 외막 단백질 H의 면역원성 검정)

  • Lee Jeong-Min
    • Microbiology and Biotechnology Letters
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    • v.34 no.3
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    • pp.273-277
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    • 2006
  • To investigate the antigenicity and protective immunity of outer membrane protein H (OmpH) in Pasteurella multocida D:4, the recombinant OmpH protein was produced in Escherichia coli. The truncated and Trx-fused form of recombinant OmpH (53 kDa) was purified, and used as an antigen in the immunization and challenge experiment. The immunized mice with the recombinant OmpH produced a high-titer antibody, and had protective immunity against P. multocida as same level as the mice immunized with formalin-killed whole cell.