Cloning, sequencing, expression, and purification of aspartic proteases isolated from two human Demodex species
Copyright © 2023 Elsevier B.V. All rights reserved..
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.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2023 |
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| Erschienen: |
2023 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:253 |
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| Enthalten in: |
International journal of biological macromolecules - 253(2023), Pt 7 vom: 31. Dez., Seite 127404 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Hu, Li [VerfasserIn] |
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| Links: |
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| Themen: |
Aspartic proteases |
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| Anmerkungen: |
Date Completed 27.11.2023 Date Revised 27.11.2023 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.ijbiomac.2023.127404 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| 500 | |a Date Revised 27.11.2023 | ||
| 500 | |a published: Print-Electronic | ||
| 500 | |a Citation Status MEDLINE | ||
| 520 | |a Copyright © 2023 Elsevier B.V. All rights reserved. | ||
| 520 | |a 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 | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Aspartic proteases | |
| 650 | 4 | |a Expression and purification | |
| 650 | 4 | |a Two human Demodex species | |
| 650 | 7 | |a Peptide Hydrolases |2 NLM | |
| 650 | 7 | |a EC 3.4.- |2 NLM | |
| 700 | 1 | |a Guan, Chenglin |e verfasserin |4 aut | |
| 700 | 1 | |a Zhao, Yae |e verfasserin |4 aut | |
| 700 | 1 | |a Zhang, Wanyu |e verfasserin |4 aut | |
| 700 | 1 | |a Chai, Rong |e verfasserin |4 aut | |
| 700 | 1 | |a Teng, Juan |e verfasserin |4 aut | |
| 700 | 1 | |a Tian, Qiong |e verfasserin |4 aut | |
| 700 | 1 | |a Xun, Meng |e verfasserin |4 aut | |
| 700 | 1 | |a Wu, Feng |e verfasserin |4 aut | |
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