• Title/Summary/Keyword: single cell MS

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Solid-phase PEGylation for Site-Specific Modification of Recombinant Interferon ${\alpha}$-2a : Process Performance, Characterization, and In-vitro Bioactivity (재조합 인터페론 알파-2a의 부위 특이적 수식을 위한 고체상 PEGylation : 공정 성능, 특성화 및 생물학적 활성)

  • Lee, Byung-Kook;Kwon, Jin-Sook;Lee, E.K.
    • KSBB Journal
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    • v.21 no.2
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    • pp.133-139
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    • 2006
  • In 'solid-phase' PEGylation, the conjugation reaction occurs as the proteins are attached to a solid matrix, and thus it can have distinct advantages over the conventional, solution-phase process. We report a case study: rhIFN-${\alpha}$-2a was first adsorbed to cation exchange resin and then N-terminally PEGylated by aldehyde mPEG of 5, 10, and 20 kD through reductive alkylation. After the PEGylation, salt gradient elution efficiently recovered the mono-PEGylate in a purified form from the unwanted species such as unmodified IFN, unreacted PEG, and others. The mono-PEGylation and its purification were integrated in a single chromatographic step. Depending on the molecular weight of the mPEG aldehyde used, the mono-PEGylation yield ranged 50-64%. We could overcome the major problems of random, or uncontrollable, multi-PEGylation and the post-PEGylation purification difficulties associated with the solution-phase process. N-terminal sequencing and MALDI-TOF MS confirmed that a PEG molecule was conjugated only to the N-terminus. Compared with the unmodified IFN, the mono-PEGylate showed the reduced anti-viral activity as measured by the cell proliferation assay. The bioactivity was reduced more as the higher molecular weight PEG was conjugated. Immunoreactivity, evaluated indirectly by antibody binding activity using a surface plasmon resonance biosensor, also decreased. Nevertheless, trypsin resistance as well as thermal stability was considerably improved.

Tissue Culture Method as a Possible Tool to Study Herbicidal Behaviour and Herbicide Tolerance Screening (조직배양(組織培養) 방법(方法)을 이용(利用)한 제초제(除草劑) 작용성(作用性) 및 제초제(除草劑) 저항성(抵抗性) 검정방법(檢定方法) 연구(硏究))

  • Kim, S.C.;Lee, S.K.;Chung, G.S.
    • Korean Journal of Weed Science
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    • v.6 no.2
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    • pp.174-190
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    • 1986
  • A series of laboratory and greenhouse experiments were conducted to find out the possibility of tissue culture and cell culture methods as a tool to study herbicidal behaviour and herbicide tolerance screening from 1985 to 1986 at the Yeongnam Crop Experiment Station. For dehulled-rice culture, pure agar medium was the most appropriate in rice growth campared to other media used for plant tissue culture method. All the media but the pure agar medium resulted in growth retardance by approximately 50% and this effect was more pronounced to root growth than shoot growth. Herbicidal phytotoxicity was enhanced under light condition for butachlor, 2.4-D, and propanil while this effect was reversed for DPX F-5384 and CGA 142464, respectively. And also, herbicides of butachlor, chlornitrofen, oxadiazon, and BAS-514 resulted in more phytotoxic effect when shoot and root of rice were exposed to herbicide than root exposure only while other used herbicides exhibited no significant difference between two exposure regimes. Similar response was obtained from Echinochloa crusgalli even though the degree of growth retardance was much greater. Particularly, butachlor, 2.4-D, chlornitrofen, oxadiaxon, pyrazolate and BAS-514 totally inhibited chlorophyll biosynthesis even at the single contact of root. Apparent cultivar differences to herbicide were observed at the young seedling culture method and dehulled rice cultivars were more tolerant in DPX F-5384, NC-311, pyrazolate and pyrazoxyfen, respectively. For derant than other types or rice cultivar in butachlor, pretilachlor, perfluidone and oxadiazon while Tongil-type rice cultivars were more tolerant in DPXF-5384, NC-311, Pyrazolate and Pyrazoxyfen, respectively. For dehulled rice culture, on the other hand, Japonica-type rice cultivar was less tolerant to herbicides of butachlor, propanil, chlornitrofen and oxadiazon that was reversed trend to young seedling culture test. Cultivar differences were also exhibited within same cultivar type. In general, relatively higher tolerant cultivars were Milyang 42, Cheongcheongbyeo, Samgangbyeo, Chilseoungbyeo for Tongil-type, Somjinbyeo for Japonica-type and IR50 for Indica-type, respectively. The response of callus growth showed similar to dehulled rice culture method in all herbicides regardless of property variables. However, concentration response was much sensitive in callus response. The concentration ranges of $10^{-9}M-10^(-8)M$ were appropriate to distinguish the difference between herbicides for E. crusgalli callus growth. Among used herbicides, BAS-514 was the most effective to E. crusgalli callus growth. Based on the above results, tissue culture method could be successfully used as a tool for studying herbicidal behaviour and tolerance screening to herbicide.

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