Expression, Purification and Biological Characteristics Prediction of Protein HmpA of Salmonella paratyphi A

Abstract

Objective: To express the protein HmpA of Salmonella paratyphi A in prokaryotes, and perform bioinformatics analysis on it to provide a theoretical reference for studying the effect of this protein on the nitric oxide (NO) signaling pathway in the host. Methods: The hmpA gene was amplified by PCR and subcloned into the T-vector. Then the expression vector pNdeI-hmpA was constructed and transformed into BL21 (DE3). After induced by IPTG, the recombinant protein expression form was assessed by SDS-PAGE. HmpA was purified using Histrap preload column and identified by Western blot. Bioinformatics technology was used to analyze the characteristics of HmpA. Results: The prokaryotic expression vector pNdeI-hmpA was successfully constructed. HmpA coexisted in the inclusion body and soluble forms under low temperatures after being induced with IPTG. The purified HmpA protein could be detected by the anti-His antibody. Bioinformatics analysis suggested that HmpA was a hydrophilic protein with no transmembrane structural domain or signal peptide. The secondary structure of this protein was mainly composed of α-helix with irregular convolutions, and the tertiary structure model was similar to a ring. The protein structural domain analysis showed that HmpA contains one functional structural domain belonging to the PRK13289 superfamily. There were 32 phosphorylation sites in HmpA. Associated with multiple Salmonella proteases or transcription factors that degrade nitrates and killer NO. Conclusion: This study successfully obtained Salmonella paratyphi A HmpA protein by genetic engineering technology and predicted part of its biological characteristics by bioinformatics methods, which would provide theoretical support for the subsequent uncovering of the influence of HmpA of Salmonella paratyphi A on the nitric oxide signaling pathway in the host.

Keywords

• salmonella paratyphi a
• prokaryotic expression
• hmpa protein
• protein purification
• bioinformatics analysis