Molecular cloning of a full-length cDNA for ECBP21 from Angelica dahurica

文献类型: 外文期刊

第一作者: Mao, GH

作者: Mao, GH;Tang, WQ;Guo, Y;Ding, CB;Zhou, RG;Sun, DY

作者机构:

关键词: extracellular calmodulin-binding protein;ECBP21;cDNA cloning;protein expression;Angelica dahurica

期刊名称:CHINESE SCIENCE BULLETIN ( 影响因子:1.649; 五年影响因子:1.738 )

ISSN: 1001-6538

年卷期: 2002 年 47 卷 13 期

页码:

收录情况: SCI

摘要: 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.

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