| Summary: | 碩士 === 國立陽明大學 === 生物藥學研究所 === 89 === The eukaryotic tRNA-modifying enzyme, tRNA-guanine transglycosylase (TGT), exchanges a guanine residue in the anticodon of tRNAs. In higher eukaryotes, the amount of the resulting queuosine nucleoside (Q) is dependent on the stage of developmental state of the respective cells. Neoplastically transformed and fast-proliferating cells usually are almost Q-deficient. During the procedures of purification of placenta TGT was quite unstable and lost its activity easily.
To overcome these difficulties, to improve the yield of the purification procedure, and to shorten the time of purification, TGT enzyme from human placenta was partially purified using TSK® HW-55 (F) column chromatography without pumping. This procedure enable the separation from IgG and hemoglobin. Then, the protein was concentrated in a Rotofor isoelectric focusing cell, specially in 2﹪glycerol and 4﹪sucrose buffer system, and subjected to IgG adsorption with Protein A Sepharose 4 Fast Flow® and GammaBind G Sephrose®. After renaturation, the enzyme was purified 4.5-fold with increase of specific activity to 784 pmol/ h-1mg-1 of protein at 37℃, pH 7.6. Using the Mini Prep Cell® method, a high degree of TGT 60 kDa subunit was obtained, and its can then be sequenced to reveal their amino acid composition or generating monoclone antibody.
Deshpande and Katze reported a search of the human EST database for sequences with significant homology to the well studied TGT from E. coli and obtained IMAGE: 72154: contains an ORF encoding a 44 kDa polypeptide. But there was no difference between differentiated or non-differentiated K562 cell in mRNA level. Without TGT activity proofing, the function of this cDNA product should be further confirmed.
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