Cloning, sequencing, expression, and purification of aspartic proteases isolated from two human Demodex species

• Published in: International journal of biological macromolecules Vol. 253; p. 127404
• Main Authors: Hu, Li, Guan, Chenglin, Zhao, Yae, Zhang, Wanyu, Chai, Rong, Teng, Juan, Tian, Qiong, Xun, Meng, Wu, Feng
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 31.12.2023
• Subjects: Aspartic proteases; Expression and purification; Two human Demodex species; Two human Demodex species; Aspartic proteases; Expression and purification
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2023.127404

Aspartic proteases (ASPs) are important hydrolases for parasitic invasion of host tissues or cells. This was the first study on Demodex ASP. First, the complete coding sequence (CDS) was amplified, cloned and sequenced. Then, the protein physical and chemical properties was analysed. Finally, the recombinant plasmid, expression and purification system was established. Results showed that the lengths of CDS of Demodex folliculorum and D. brevis were 1161 and 1173 bp, respectively. The molecular weight of the protein was approximately 40 KDa. It contained an aspartic acid residue, a substrate-binding site and signal peptide, yet lacked a transmembrane domain and was located in the membrane or extracellular matrix. The phylogenetic and conserved motif analyses showed that D. folliculorum and D. brevis clustered separately and then formed a single branch, which finally clustered with other Acariformes species. The prokaryotic expression systems for recombinant ASP with His-tag (rASP-His) and GST-tag (rASP-GST) were constructed. The inclusion bodies of rASP-His were renaturated by gradient urea and purified using NI beads, while those of rASP-GST were renaturated by sarkosyl and Triton X-100 and purified using GST beads. Conclusively, the prokaryotic expression and purification system of Demodex rASP was successfully established for further pathogenic mechanism research. •The first study on Demodex ASP gene amplification, expression and purification.•ASP CDSs were successfully amplified by OE-PCR from the two human Demodex species.•ASP of two species predicted to be a secretory protein with similar conserved motifs.•Modified urea gradient and S-T methods were suitable to renaturate inclusion bodies.•It provides a basis for study on molecular pathogenic mechanism of ASP in Demodex.