Chinese Science Bulletin

, 47:1100 | Cite as

Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

  • Guohong Mao
  • Wenqiang Tang
  • Yi Guo
  • Cunbao Ding
  • Rengang Zhou
  • Daye Sun
Notes
  • 13 Downloads

Abstract

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca2+-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.

Keywords

extracellular calmodulin-binding protein ECBP21 cDNA cloning protein expression Angelica dahurica 

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Copyright information

© Science in China Press 2002

Authors and Affiliations

  • Guohong Mao
    • 1
  • Wenqiang Tang
    • 1
  • Yi Guo
    • 1
  • Cunbao Ding
    • 1
  • Rengang Zhou
    • 2
  • Daye Sun
    • 1
  1. 1.Institute of Molecular Cell BiologyHebei Normal UniversityShijiazhuangChina
  2. 2.Institute of Agro-physics, Physiology and BiochemistryAcademy of Agricultural ScienceShijiazhuangChina

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