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

Abstract

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.

Keywords: Arca inflata; In vitro anti-tumor activity; Protein; Purification; Structural characterization.