Characterization of Thermostable Deblocking Aminopeptidases of Archaeon Thermococcus onnurineus NA1 by Proteomic and Biochemical Approaches.



Yeol Gyun Lee1, Sun-Hee Leem2,Young-Ho Chung1, and Seung Il Kim1


  1. Division of Life Science, Korea Basic Science Institute, Daejeon 305-333, Republic of Korea
  2. Department of Biology, Dong-A University, Busan 602-715, Republic of Korea


Thermococcus onnurineus NA1 is a hyperthermophilic archaeon that grows optimally at >80°C. The deblocking aminopeptidase (DAP) (TNA1-DAP1) encoded in Ton_1032 of T. onnurineus NA1 is considered a major DAP. However, four genes encoding putative DAP have been identified from a genomic analysis of T. onnurineus NA1. A proteomic analysis revealed that all four DAPs were differentially induced in YPS culture medium and, particularly, two DAPs (TNA1-DAP1 and TNA1-DAP2) were dominantly expressed in T. onnurineus NA1. The biochemical properties and enzyme activity of DAPs induced in an E. coli expression system suggested that the two major DAPs play complementary roles in T. onnurineus NA1.

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The deblocking aminopeptidase (DAP) (TNA1-DAP1) encoded in Ton_1032 of T.onnurineus NA1 was identified, cloned, and expressed before recombinant DAPs were purified. Equal volumes of protein solution and sinapic acid (10 mg/ml in 50% CAN/0.1% TFA) were mixed before 1 μL of the mixture was spotted onto a MALDI plate and dried at room temperature. Prior to analysis, the instrument was calibrated using the CovalX C150 and C450 calibration kits. The plate was then analyzed on a mass spectrometer that had been modified with a CovalX HM2 detection system. The data obtained from the analysis was analyzed using the CovalX Complex Tracker software.



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