곤충세포 배지로부터 히스티딘이 융합된 Autotaxin(NPP-2)의 발현, 분비 및 정제

• 영문명: Expression, Secretion and Purification of Histidine-Tagged Autotaxin (NPP2) from Insect Cells Media
• 발행기관: 대한약학회
• 저자명: 이종한(Jong Han Lee) 송재휘(Jaehwi Song) 이종흔(Jong Heun Lee) 안영민(Young Min Ahn) 김수영(Su Young Kim) 이석형(Sug Hyung Lee) 박원상(Won Sang Park) 유남진(Nam Jin Yoo) 홍성렬(Sung Yeol Hong) 이정용(Jung Young Lee) 남석우(Suk Woo Nam)
• 간행물 정보: 『약학회지』제47권 제6호 (2003년), 410~416쪽, 전체 7쪽
• 주제분류: 의약학 > 기타의약학
• 파일형태: PDF
• 발행일자: 2003.12.31

영문 초록

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 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 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.

키워드

autotaxin purification insect cell.