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Purification and structural characterization of a novel anti-tumor protein from Arca inflata

Zhu, Jianhua, Xu, Jian, Wang, Yanqing, Li, Chunlei, Chen, Zhiyan, Song, Liyan, Gao, Jingjing, Yu, Rongmin
International journal of biological macromolecules 2017
Anadara broughtonii, Crassostrea gigas, Fourier transform infrared spectroscopy, absorption, antineoplastic activity, carbohydrate content, circular dichroism spectroscopy, databases, gel electrophoresis, glycoproteins, humans, inhibitory concentration 50, isoelectric point, molecular weight, neoplasm cells, reversed-phase high performance liquid chromatography, sequence analysis, transcriptome, triose-phosphate isomerase, unigenes
A novel in vitro anti-tumor protein (J2-C2) with a molecular weight of 27,153.0Da was isolated from the edible portion of Arca inflata. Physical and structural properties of J2-C2 were characterized using physicochemical and instrumental analyses. Gel electrophoresis analysis showed that J2-C2 is a homogeneous, monomeric protein with an isoelectric point of 6.3. The purity of the isolated native J2-C2 was >99%, as determined by RP-HPLC. The carbohydrate content assay showed that J2-C2 was not a glycoprotein. The FT-IR spectrum of J2-C2 gave characteristic amide absorption bands at 1645.71 and 1541.46cm−1. Secondary structure analysis by CD spectroscopy revealed that J2-C2 had 34.0% α-helix, 27.5% β-sheet, 13.4% β-turn and 25.1% random coil. In-gel and nano ESI-MS/MS sequencing analysis combined with transcriptome unigene analysis yielded the complete amino acid sequence of J2-C2. Aligning with NCBI BLAST database, J2-C2 showed 77% homology with predicted triosephosphate isomerase (TIM) derived from Crassostrea gigas. Therefore, J2-C2 was considered to be a new TIM-like protein in A. inflata. The anti-tumor effect of J2-C2 against three human tumor cells was measured by MTT assay, and the IC50 values of J2-C2 were 42.38, 45.64 and 48.73μM against A549, HepG2 and SPC-A-1 cell lines, respectively.