Bioinformatics analysis,eukaryotic expression and mass spectrum identification of the tilapia HSP70 protein

The physical and chemical property, glycosylation site, transmembrance domain, cellular localization, signal peptide of tilapia HSP70(tHSP70)and percent identity to the HSP70 derived from rainbow trout and other species(tHSP70)were analyzed by bioinformatics methods, and the full CDS of tHSP70 was amplified and cloned by using and the artificially synthesized cDNA. The expression plasmid of pGAPZa-HSP70 was constructed by the insertion of tHSP70 CDS into the pGAPZa-A vector, and the tHSP70 protein was then expressed in host strain of GS115. The supernatant was used to conduct the SDS-PAGE and Western-blotting, followed by the purification of tHSP70 by Ni₂⁺IDA affinity chromatography. Then the PAS staining was used to identify if the glycosylations occurred in tHSP70 protein after the expression from GS115. Then the non-expected band(about 100 bp)in the SDS-PAGE was identified by standard MALDI-TOF-MS procedure. Bioinformatic analysis showed tHSP70 contains 640 amino acids, the molecular weight is 70 274.5 mol/L and the isoelectric point is 5. 49. The half-life of tHSP70 is 30 h in mammalian reticulocyte(in vitro)and greater than 20 h in yeast, while it is more than 10 h in Escherichia coli. Additionally, the instability index and aliphatic index of tHSP70 is 36. 64 and 85. 58, respectively. Moreover, tHSP70 might totally contain 6 O-linked and 6 N-linked glycosylation sites. The cloned tHSP70 sequence was 100% identity to the sequence in NCBI database. The HSP70 protein could be efficiently expressed by methane induction of pGAPZa-HSP70 plasmid in GS115 strain. Except the 70 ku band, one 100 ku was observed on the SDS-PAGE and Western blotting gels. The PAS staining specific to the glycosyl radicals was performed and the positive signals indicated that this 100 ku band might be the consequence of glycosylations after expression in GS115. Western blotting showed the protein currently expressed was recognized by rabbit anti-human HSP70 antibody, and the MALDI-TOF-MS result demonstrated that the 100 ku protein was exactly the tilapia HSP70, 100% identity to the tilapia HSP70 in NCBI database. The present study provided experimental materials for the future cellular and immunological researches in tilapia.