Purification and characterization of His-tag L-proline dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus

LIN LeYi1; ZHANG LiYe1; Shin-ichiro SUYE2; ZHENG HaiTao3; Satomi SHIRAISHI2; Yosuke OKEZAKI2

Journal of Beijing University of Chemical Technology ›› 2010, Vol. 37 ›› Issue (6) : 121-125.

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Journal of Beijing University of Chemical Technology ›› 2010, Vol. 37 ›› Issue (6) : 121-125.
生物技术与环境工程

Purification and characterization of His-tag L-proline dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus

  • LIN LeYi1; ZHANG LiYe1; Shin-ichiro SUYE2; ZHENG HaiTao3; Satomi SHIRAISHI2; Yosuke OKEZAKI2
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Abstract

A His-tag marked gene fragment of dye-linked L-proline dehydrogenase (dye-linked L-pro DH) from a hyperthermophilic archaeon, Thermococcus profundus, has been used as the target gene to express the desired protein with Escherichia coli Rosetta-gami (DE3) competent cells. The His-tag L-pro DH was gained through fermentation, and purified by using cell-free extraction, heat treatment and Ni Sepharose chromatography, resulting in a five-fold increase in specific activity. Three peptides with molecular weights of 58600, 40200 and 22300 were obtained. Preliminary experiments suggested that the maximum enzyme activity of His-tag L-pro DH, 1.5 U/mL, was obtained under the following conditions: 50 ℃, 300 mmol/L Tris buffer at a pH of 8.0.

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LIN LeYi1; ZHANG LiYe1; Shin-ichiro SUYE2; ZHENG HaiTao3; Satomi SHIRAISHI2; Yosuke OKEZAKI2. Purification and characterization of His-tag L-proline dehydrogenase from a hyperthermophilic archaeon, Thermococcus profundus[J]. Journal of Beijing University of Chemical Technology, 2010, 37(6): 121-125

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