Establishment of purification method for prokaryotic expression of Serpin gene for Dermatophagoides farinae
Copyright © 2022 Elsevier Inc. All rights reserved..
This study aimed to develop an effective method for the expression and purification of the Dermatophagoides farinae serpin protein and to establish an experimental foundation for elucidating its role in the temperature stress response. The total RNA of D. farinae was extracted, and specific primers were designed for serpin amplification. Serpin was joined with pET32a vector and transformed into BL21 (DE3) cells. Expression of recombinant proteins was induced. Proteins were extracted by enzymatic lysis or enzymatic lysis combined with ultrasonication. Recombinant proteins were purified by Ni-NTA method. SDS-PAGE was conducted to evaluate protein expression, extraction, and purification efficiency. Agarose gel electrophoresis and sequencing analysis showed that the amplified serpin open reading frame was 1284 bp, encoding a hydrophilic and stable protein with a relative molecular weight of 48.30 kD. SDS-PAGE demonstrated that there was a specific band at 55-70 kD, which was consistent with the predicted size of the recombinant pET32a-Serpin protein. Enzymatic lysis combined with 30% ultrasonic power promoted the release of soluble protein more effectively than enzymatic lysis alone. 16 °C for 4 h was optimal for inducing expression. The optimal imidazole concentrations for washing non-His-tagged protein and eluting His-tagged protein were determined to be 20 mM and 200 mM, respectively. In this study, A prokaryotic expression and purification system for the D. farinae serpin protein was successfully established, providing a technical reference for functional gene research in mites at the protein level.
| Medienart: |
E-Artikel |
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| Erscheinungsjahr: |
2022 |
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| Erschienen: |
2022 |
| Enthalten in: |
Zur Gesamtaufnahme - volume:195-196 |
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| Enthalten in: |
Protein expression and purification - 195-196(2022) vom: 01. Aug., Seite 106080 |
| Sprache: |
Englisch |
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| Beteiligte Personen: |
Zhang, Wanyu [VerfasserIn] |
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| Links: |
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| Themen: |
Dermatophagoides farinae |
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| Anmerkungen: |
Date Completed 04.05.2022 Date Revised 14.05.2022 published: Print-Electronic Citation Status MEDLINE |
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| doi: |
10.1016/j.pep.2022.106080 |
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| funding: |
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| Förderinstitution / Projekttitel: |
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| PPN (Katalog-ID): |
NLM338348085 |
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| 520 | |a Copyright © 2022 Elsevier Inc. All rights reserved. | ||
| 520 | |a This study aimed to develop an effective method for the expression and purification of the Dermatophagoides farinae serpin protein and to establish an experimental foundation for elucidating its role in the temperature stress response. The total RNA of D. farinae was extracted, and specific primers were designed for serpin amplification. Serpin was joined with pET32a vector and transformed into BL21 (DE3) cells. Expression of recombinant proteins was induced. Proteins were extracted by enzymatic lysis or enzymatic lysis combined with ultrasonication. Recombinant proteins were purified by Ni-NTA method. SDS-PAGE was conducted to evaluate protein expression, extraction, and purification efficiency. Agarose gel electrophoresis and sequencing analysis showed that the amplified serpin open reading frame was 1284 bp, encoding a hydrophilic and stable protein with a relative molecular weight of 48.30 kD. SDS-PAGE demonstrated that there was a specific band at 55-70 kD, which was consistent with the predicted size of the recombinant pET32a-Serpin protein. Enzymatic lysis combined with 30% ultrasonic power promoted the release of soluble protein more effectively than enzymatic lysis alone. 16 °C for 4 h was optimal for inducing expression. The optimal imidazole concentrations for washing non-His-tagged protein and eluting His-tagged protein were determined to be 20 mM and 200 mM, respectively. In this study, A prokaryotic expression and purification system for the D. farinae serpin protein was successfully established, providing a technical reference for functional gene research in mites at the protein level | ||
| 650 | 4 | |a Journal Article | |
| 650 | 4 | |a Research Support, Non-U.S. Gov't | |
| 650 | 4 | |a Dermatophagoides farinae | |
| 650 | 4 | |a Ni-NTA purification | |
| 650 | 4 | |a Prokaryotic expression | |
| 650 | 4 | |a Serpin | |
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| 650 | 7 | |a Serpins |2 NLM | |
| 700 | 1 | |a Zhao, Yae |e verfasserin |4 aut | |
| 700 | 1 | |a Hu, Li |e verfasserin |4 aut | |
| 700 | 1 | |a Guan, Chenglin |e verfasserin |4 aut | |
| 700 | 1 | |a Xun, Meng |e verfasserin |4 aut | |
| 700 | 1 | |a Wu, Feng |e verfasserin |4 aut | |
| 700 | 1 | |a Lei, Yanjun |e verfasserin |4 aut | |
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