Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica |
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Authors: | Guohong Mao Wenqiang Tang Yi Guo Cunbao Ding Rengang Zhou Daye Sun |
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Affiliation: | (1) Institute of Molecular Cell Biology, Hebei Normal University, 050016 Shijiazhuang, China;(2) Institute of Agro-physics, Physiology and Biochemistry, Academy of Agricultural Science, 050051 Shijiazhuang, China |
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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. |
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Keywords: | extracellular calmodulin-binding protein ECBP21 cDNA cloning protein expression Angelica dahurica |
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