The recombinant protein,Hq001,in Haemaphysalis qinghaiensis:Structure prediction and prokaryotic expression
Objective To construct the three-dimensional theoretical model of Hq001 protein encoded by a novel gene,Hq001,cloned from a salivary gland cDNA library of Haemaphysalis qinghaiensis,and to evaluate its relationship with tick-derived Kunitz-type anticoagulant proteins.Methods The recombinant plasmid pET-30a-Hq001 was constructed and transformed into Escherichia coli BL21(DE3)for the expression of Hq001 protein.AlphaFold v2 software was used to predict the tertiary structure of Hq001 amino acid sequence with the signal peptide removed,to obtain a three-dimensional(3D)theoretical model.The 3D theoretical model was optimized through molecular dynamics simulation using the open-source software GROMACS v2023 and evaluated using ANOLEA and MolProbity.Results The recombinant plasmid pET30a-Hq001 was expressed as inclusion bodies in E.coli BL21.Sequence alignment and structure modeling results showed that Hq001 protein had typical double Kunitz-BPTI domains,with a similar sequence and structure to four Kunitz-BPTI-type anticoagulant proteins,such as Bikunin,Boophilin,Ornithodorin,and Ixolaris.Following 1 ns molecular dynamics(MD)simulation optimization,the root mean square deviation(RMSD)of the theoretical model attained stability at approximately 3 A,indicative of convergence to a steady-state conformational ensemble.Concurrently,the most energetically favorable structure within this ensemble,exhibiting a total energy of-207 166 400 kJ/mol,was identified and designated as the final model.Conclusion Hq001 protein has conserved Kunitz-BPTI domains that resemble Boophilin from Rhipicephalus microplus in sequence and structure,indicating that Hq001 protein may have similar structure and function to some known Kunitz-type serine protease inhibitors.
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