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Abstract <p>This paper reports the design of a thermostable temperature-activated mini-intein based on the full-length intein DnaE1 from <i>Thermus thermophilus</i> HB27 (<i>Tth</i>DnaE1). We performed rational design of three mini-inteins <i>Tth</i>DnaE1 Δ272, Δ280, and Δ287 through deletion mutations in the full-length intein sequence. Two mini-inteins (Δ272 and Δ280) were capable of efficient protein splicing at temperatures above 50°C. The most active mini-intein with the Δ280 deletion was selected as a platform for further design of a self-cleaving carrier of affinity tags through single-point mutagenesis. Three mutations – C1A, D405G, and the combined C1A/D405G – were introduced to inhibit N-terminal extein cleavage and extein ligation. As a result, the mini-intein Δ280 with double mutation C1A/D405G displayed the highest efficiency of C-terminal extein cleavage with temperature optimum around 60°C. Thus, we constructed thermostable temperature-activated mini-intein capable of efficient protein splicing or cleavage of the C-terminal extein. The engineered <i>Tth</i>DnaE1 Δ280 C1A/D405G mini-intein can serve as a basis for the development of new expression system for intein-mediated production of pharmaceutically relevant recombinant proteins and peptides.</p>

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Designing a Thermostable Mini-Intein for Intein-Mediated Purification of Recombinant Proteins and Peptides

  • 期刊论文

  • 发表日期：

    2025年6月1日

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  • Andrey A. Karanov,
  • Evgeniy A. Zayats,
  • Maria A. Kostromina,
  • Yulia A. Abramchik,
  • Aleksandra R. Sharafutdinova,
  • Maria S. Surkova,
  • Andrey A. Zamyatnin Jr.,
  • Roman S. Esipov

摘要

Abstract

This paper reports the design of a thermostable temperature-activated mini-intein based on the full-length intein DnaE1 from Thermus thermophilus HB27 (TthDnaE1). We performed rational design of three mini-inteins TthDnaE1 Δ272, Δ280, and Δ287 through deletion mutations in the full-length intein sequence. Two mini-inteins (Δ272 and Δ280) were capable of efficient protein splicing at temperatures above 50°C. The most active mini-intein with the Δ280 deletion was selected as a platform for further design of a self-cleaving carrier of affinity tags through single-point mutagenesis. Three mutations – C1A, D405G, and the combined C1A/D405G – were introduced to inhibit N-terminal extein cleavage and extein ligation. As a result, the mini-intein Δ280 with double mutation C1A/D405G displayed the highest efficiency of C-terminal extein cleavage with temperature optimum around 60°C. Thus, we constructed thermostable temperature-activated mini-intein capable of efficient protein splicing or cleavage of the C-terminal extein. The engineered TthDnaE1 Δ280 C1A/D405G mini-intein can serve as a basis for the development of new expression system for intein-mediated production of pharmaceutically relevant recombinant proteins and peptides.