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Expression of flounder Paralichthys olivaceus calmodulin gene in prokaryotic and eukaryotic cells

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Author(s): LÜ Ai-Jun | HU Xiu-Cai

Journal: Acta Zoologica Sinica
ISSN 0001-7302

Volume: 54;
Issue: 6;
Start page: 1061;
Date: 2008;
Original page

Keywords: Flounder | Calmodulin gene | Prokaryotic expression | Eukaryotic expression

ABSTRACT
The primer was designed based on the sequence which was obtained from subtractive cDNA library, and then cloned the flounder Paralichthys olivaceus calmodulin gene(PoCaM) using SMART cDNA as the model. Computer-assisted analysis revealed the potential open reading frame encoded a protein of 149 amino acids with a predicted size of 17 kDa, the predicted theoretical isoelectric points (PI) is 3.93. Sequence alignment of PoCaM with other well known calmodulin proteins showed a significant homology with identity of 97.3%–100%, which contained helix-loop-helix motif. The recombinant expressing vector pET32a/PoCaM was successfully constructed and transformed into the bacteria BL21 (DE3) cells which were then induced with isopropyl-1-thio-b-D-galactopyranoside (IPTG). SDS-PAGE analysis showed that the recombinant E.coli could express a 34 kD protein which is consistent with the size of estimated pET32a/PoCaM fusion protein. The transient expression of recombinant pEGFP-N3/PoCaM in EPC (Epithelioma papulosum cyprinid)cells showed that PoCaM distributed in nucleus and cytoplasm under fluorescence microscopy[Acta Zoologica Sinica 54(6): 1061– 1067, 2008].
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