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KMID : 0370220030470060410
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Yakhak Hoeji
2003 Volume.47 No. 6 p.410 ~ p.416
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Expression, Secretion and Purification of Histidine-Tagged Autotaxin (NPP2) from Insect Cells Media
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이종한/Lee JH
송재휘/이종흔/안영민/김수영/이석형/박원상/유남진/Song JH/Lee JH/Ahn YM/Kim SY/Lee SH/Park WS/Yoo NJ
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Abstract
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Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulting factor, possessing a type II nucl eotide pyrophosphatase/phosphodiesterase (NPP2) activity. Recombinant ATX has recently demonstrated that human plasma lysophosholipase D is identical to ATX and uses lysophosphatidylcholine as a substrate to mediate various biological functions including tumor cell growth and motility through G-protein coupled receptor. However, despite pivotal roles of ATX on physiological or pathophysiological states, the production of ATX is solely depends on complicated purification method which employs multiple column steps, but resulted in very poor yield. This limited the use of ATX for extensive analysis. We, therefore, expressed six histidine-tagged recombinant human ATX(His-ATX) in High FiveTM insect cells to improve the generation of ATX and to make simple the purification of ATX. The signal sequence of the human ATX gene was truncated and replaced with sequence of insect cell secretion signal within expression vector. In addition, codons for six histidines were added to the C- termini of 120kDa ATX cDNA construct. A simple purification scheme utilizing two-step affinity column chromatography was designed to purify His-ATX to homogeneity from the culture supernatant of transfected insect cells. Homogenous His-ATX was detected and isolated from the concentrated insect cell medium using concanavalin A agarose and nickel affinity chromatography. Purified His-ATX was in full length with ATX capacity. A combination of this expression system and purification scheme would be usedful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.
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