Sequence analysis, prokaryotic expression and purification of Salmonella typhimurium Ssek3 protein / 中华微生物学和免疫学杂志

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 ob-tained from Salmonella typhimurium SL1344 strain.Bioinformatics methods were used for systematic analy-sis .A prokaryotic expression system for expressing Sse3k gene was constructed and the expressed protein was purified by Ni-NTA affinity chromatography .Results Sequence analysis showed that the Ssek3 gene of Sal-monella typhimurium was 1008 bp in length, encoding a protein of 335 amino acids and 72 amino acid resi-dues.The molecular weight, molecular formula and isoelectric point of Ssek3 protein was 37.89×103, C1700 H2629 N463 O497 S12 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 di-sulfide bonds.The secondary structure of Ssek3 protein contained 114 α-helices (Hh) (34.03％), 72 ex-tended chain (Ee) (21.49％), 30β-sheets (Tt) (8.96％) and 119 random coils (Cc) (35.52％).Re-sults of SDS-PAGE showed that the fusion protein Ssek 3 expressed in the prokaryotic expression system was a secretory protein with a molecular weight of about 40×103 .Conclusion The Ssek3 gene of Salmonella typh-imurium is successfully cloned , sequenced and expressed in this study , which will lay a foundation for fur-ther studying the role of Ssek3 protein in host cells during Salmonella typhimurium infection.