Prediction of an Efficient Signal Peptide for Optimized Expression of Mycobacterium Tuberculosis Heparin-binding Hemagglutinin Gene in Periplasmic Compartment of Escherichia Coli: A Bioinformatics Investigation

Background: The heparin-binding hemagglutinin (HBHA) protein belonging to Mycobacterium tuberculosis is known as a molecular adjuvant.  Objective: Hence, the expression of this protein in the prokaryotic system is essential. Methods: To predict an appropriate signal peptide for the expression of the HBHA protein, 50 signal peptides were selected from the signal peptide database. Then, the crucial parameters of signal peptides, including the probability of signal peptide, different regions of signal peptides, physicochemical features, sorting of signal peptides, and sub-cellular location were completely investigated by reliable tools. After the best-predicted signal peptide was identified, it was linked to the HBHA protein, and its secondary structure, tertiary structure, and in silico cloning in pET21a (+) was assessed.  Findings: The results of different evaluations confirmed that only 13 signal peptides passed all features, including clearance of N, H, and C regions, D-score >0.7, instability index >40, and periplasmic localization. Finally, based on D-scores, among these 13 signal peptides, the asr (acid shock RNA) peptide with D-score=90 was selected as the best-predicted signal peptide to apply. Moreover, the results showed that the secondary structure of the adjuvant linked to asr peptide contained 88.18% alpha helix and 9.5% random coil. Also, the results of in silico cloning showed that the nucleotide sequences of the adjuvant linked to the asr peptide were successfully cloned between BamHI and XhoI enzymes in the multiple cloning site of pET21a (+). Conclusion: The results of this study confirmed that the asr peptide can be used as an appropriate signal peptide for the expression of the HBHA protein in the prokaryotic system.