YAKHAK HOEJI (약학회지)

The Pharmaceutical Society of Korea (대한약학회)
권호 표지

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

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

  • 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)
  • Published : 2003.12.01
Download PDF
⟨ Previous Next ⟩

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

References

  1. Stracke, M. L., Krutzsch, H. C., Unsworth, E. J., Arestad, A., Cioce, V., Schiffmann, E. and Liotta, L. A. : Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein. J Biol. Chem. 267(4), 2524 (1992)
  2. Murata, J., Lee, H. Y., Clair, T., Krutzsch, H. C., Arestad, A. A., Sobel, M. E., Liotta, L. A. and Stracke, M. L. : eDNA cloning of the human tumor motility-stimulating protein, autotaxin, reveals a homology with phosphodiesterases. J. Biol. Chem. 269(48), 30479 (1994)
  3. Clair, T., Lee, H. Y., Liotta, L. A. and Stracke, M. L. : Autotaxin is an exoenzyme possessing 5'-nucleotide phosphodiesterase/ATP pyrophosphatase and ATPase activities. J Biol. Chem. 272(2), 996 (1997)
  4. Mulvaney, P. T., Stracke, M. L., Nam, S. W., Woodhouse, E., O'Keefe, M., Clair, T., Liotta, L. A., Khaddurah-Daouk, R. and Schiffmann, E. : Cyclocreatine inhibits stimulated motility in tumor cells possessing creatine kinase. Int. J, Cancer. 78(1),46 (1998)
  5. Nam, S. W., Clair, T., Campo, C. K., Lee, H. Y., Liotta, L. A. and Stracke, M. L. : Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cells. Oncogene 19, 241 (2000) https://doi.org/10.1038/sj.onc.1203263
  6. Nam, S. W., Clair, T., Kim, Y. S., McMarlin, A., Schiffmann, E., Liotta, L. A. and Stracke, M. L. : Autotaxin (NPP-2), a metastasis-enhancing motogen, is an angiogenic factor. Cancer Res. 61, 6938 (2001)
  7. Bachner, D., Ahrens, M., Betat, N., Schroder, D. and Gross, G. : Developmental expression analysis of murine autotaxin (ATX). Mech. Dev., 84, 121 (1999)
  8. Fuss, B., Baba, H., Phan, T., Tuohy, V. K. and Macklin, W. B. : Phosphodiesterase I, a novel adhesion molecule and/or cytokine involved in oligodendrocyte function. J. Neurosci. 17, 9095 (1997)
  9. Umezu-Goto, M., Kishi, Y., Taira, A., Hama, K., Dohmae, N., Takio, K., Yamori, T., Mills, G. B., Inoue, K., Aoki, J. and Arai, H. : Autotaxin has lysophospholipase D activity leading to tumor cell growth and motility by lysophosphatidic acid production. J. Cell Biol. 158, 227 (2002) https://doi.org/10.1083/jcb.200204026
  10. Tokumura, A., Majima, E., Kariya, Y., Tominaga, K., Kogure, K., Yasuda, K. and Fukuzawa, K. : Identification of human plasma lysophospholipase D, a lysophosphatidic acid-producing enzyme, as autotaxin, a multifunctional phosphodiesterase. J. Biol. Chem. 277, 39436 (2002) https://doi.org/10.1074/jbc.M205623200
  11. Mills, G. B. and Moolenaar, W. H. : The emerging role of lysophosphatidic acid in cancer. Nat. Rev. Cancer 3(8), 582 (2003)
  12. Nam, S. W., Clair, T., Schiffmann, E., Liotta, L. A. and Stracke, M. L. : A sensitive screening assay for secreted motility-stimulating factors. Cell Motil. Cytoskelet. 46, 279 (2000) https://doi.org/10.1002/1097-0169(200008)46:4<279::AID-CM5>3.0.CO;2-P
  13. Stracke, M. L., Clair, T. and Liotta, L. A. : Autotaxin, tumor motility-stimulating exophosphodiesterase. Adv. Enzyme Regul. 37, 135 (1997) https://doi.org/10.1016/S0065-2571(96)00017-9
  14. Clair, T., Aoki, J., Koh, E., Bandle, R. W, Nam, S. W., Ptaszynska, M. M., Mills, G. B., Schiffmann, E., Liotta, L. A. and Stracke, M. L. : Autotaxin hydrolyzes sphingosylphos-phorylcholine to produce the regulator of migration, sphingosine-1-phosphate. Cancer Res. 63(17), 5446 (200)