수산해양교육연구 (Journal of Fisheries and Marine Sciences Education)

  • 제28권4호
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  • Pages.903-912
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  • 2016
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  • 1229-8999(pISSN)
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  • 2288-2049(eISSN)
한국수산해양교육학회 (The Korean Society for Fisheries and Marine Sciences Education)
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먹장어 Cathepsin L의 분자생물학적 클로닝, 발현 및 효소학적 특성 분석

Cloning, Expression Analysis and Enzymatic Characterization of Cathepsin L from the Inshore Hagfish (Eptatretus burgeri)

  • 투고 : 2016.03.21
  • 심사 : 2016.05.30
  • 발행 : 2016.08.31
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초록

Hagfish which belongs to the chordate contact cyclostomata, is important phylogenetic relationship between vertebrate and invertebrate. Cathepsins of the cysteine protease family have traditionally been thought to play a major role in intracellular protein degradation and turnover in lysosomes. In this study, Catepsin L was cloned from Inshore hagfish (Eptatretus burgeri), the cDNA encoding ORF of the Eptatretus burgeri Cathepsin L (EbCtL) is 978 bp. The cDNA encoding proEbCtL was expressed in Escherichia coli strain BL21(DE3) using the pGEX-4T-1 expression vector system. The recombinant proEbCtL protein was overexpressed as a approximately 55 kDa fusion protein. The overproduced soluble GST-fusion protein was then applied to glutathione-Sepharose 4B column chromatography; the sample harboring the fusion protein evidenced a high degree of purity when analyzed via SDS-PAGE and Western blot analysis. Its activity was quantied by cleaving the synthetic peptide Z-FR-AMC, Z-LLE-AMC, and Suc-AAF-AMC, and the optimal pH for the protease activity was 8, 9.5, and 9, respectively.

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