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Expression, Secretion and Purification of Histidine-Tagged Autotaxin (NPP2) from Insect Cells Media  

이종한 (가톨릭대학교 의과대학 병리학교실)
송재휘 (성균관대학교 자연과학대학 유전공학과)
이종흔 (가톨릭대학교 의과대학 병리학교실)
안영민 (경희대학교 의과대학 한의과대학)
김수영 (가톨릭대학교 의과대학 병리학교실)
이석형 (가톨릭대학교 의과대학 병리학교실)
박원상 (가톨릭대학교 의과대학 병리학교실)
유남진 (가톨릭대학교 의과대학 병리학교실)
홍성렬 (성균관대학교 자연과학대학 유전공학과)
Publication Information
YAKHAK HOEJI / v.47, no.6, 2003 , pp. 410-416 More about this Journal
Abstract
Autotaxin(ATX) was originally purified from conditioned media of A2058 human melanoma cells and shown to be a potent cell motility-stimulating factor, possessing a type II nucleotide 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 Five TM 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 useful for production and purification of high-quality functional ATX for research and practical application of multiple functional motogen, ATX/NPP-2.
Keywords
autotaxin; purification; insect cell;
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