Molecular characterization of a profilin gene from a parasitic ciliate Cryptocaryon irritans.

ABSTRACT

Profilin, known as one of the core actin-binding proteins, is an integral part of actin-based cytoskeleton involved in cell motility, cytokinesis, neuronal differentiation, and synaptic plasticity. In this study, a putative profilin gene designated as CiProfilin (GenBank accession number JX987286) was screened out from a cDNA library of Cryptocaryon irritans trophonts. The full-length cDNA of CiProfilin gene is 582 bp, containing an open reading frame (ORF) of 471 bp, which encodes a polypeptide consisting of 156 amino acids with a predicted molecular weight of 17.3 kDa. Quantification of CiProfilin mRNA expression by real-time PCR suggested that CiProfilin was expressed in all stages of C. irritans life cycle with a significantly higher level in trophonts. Five non-universal codons (TAAs) coding glutamines (Gln) were found in the ORF and mutated to CAAs (universal codons for Gln) by site-directed mutagenesis. Then the modified ORF was inserted into the plasmid pGEX-4T-1, the recombinant plasmid was subsequently transformed into Escherichia coli. The bacteria were subsequently induced to express the recombinant CiProfilin protein fused with glutathione S transferase (G-rCiProfilin), which was then purified with glutathione sepharose 4B and thrombin cleavage. The molecular weight and the antigenicity of rCiProfilin were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. The native CiProfilin was found abundant in the peripheral area beneath the cell membrane and around the cytostomes of theronts, suggesting its vital roles in food uptake, stomatogenesis, and parasitic invasion. Co-precipitation assay also revealed the activity of rCiProfilin in actin binding. This study will help further elucidate the specific roles of CiProfilin on the growth of C. irritans and the preliminary mechanism of its invasion to hosts.