Sequence analysis, prokaryotic expression and purification of Salmonella typhimurium Ssek3 protein

Abstract

Objective To study the sequence structure of Salmonella typhimurium Ssek3 gene and to express it at protein level in a prokaryotic expression system. Methods Sequence of Ssek3 gene was obtained from Salmonella typhimurium SL1344 strain. Bioinformatics methods were used for systematic analysis. A prokaryotic expression system for expressing Ssek3 gene was constructed and the expressed protein was purified by Ni-NTA affinity chromatography. Results Sequence analysis showed that the Ssek3 gene of Salmonella typhimurium was 1 008 bp in length, encoding a protein of 335 amino acids and 72 amino acid residues. The molecular weight, molecular formula and isoelectric point of Ssek3 protein was 37.89×103, C1700H2629N463O497S12 and 6.7, which indicated that it was a stable and hydrophilic protein. Ssek3 protein was a membrane protein without signal peptide or transmembrane region, containing five N-glycosylation sites, three O-glycosylation sites, 33 phosphorylation sites, 22 linear B-cell epitopes, 11 T-cell epitopes and 21 disulfide bonds. The secondary structure of Ssek3 protein contained 114 α-helices (Hh) (34.03%), 72 extended chain (Ee) (21.49%), 30 β-sheets (Tt) (8.96%) and 119 random coils (Cc) (35.52%). Results of SDS-PAGE showed that the fusion protein Ssek3 expressed in the prokaryotic expression system was a secretory protein with a molecular weight of about 40×103. Conclusion The Ssek3 gene of Salmonella typhimurium is successfully cloned, sequenced and expressed in this study, which will lay a foundation for further studying the role of Ssek3 protein in host cells during Salmonella typhimurium infection. Key words: Salmonella typhimurium; Ssek3 gene; Clone; Prokaryotic expression; Bioinformatics analysis