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

---

Andrey A. Karanov^1,2,a*, Evgeniy A. Zayats¹, Maria A. Kostromina¹, Yulia A. Abramchik¹, Aleksandra R. Sharafutdinova¹, Maria S. Surkova¹, Andrey A. Zamyatnin, Jr.^2*, and Roman S. Esipov^1,2,b

¹Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

²Faculty of Bioengineering and Bioinformatics, Lomonosov Moscow State University, 119234 Moscow, Russia

^* To whom correspondence should be addressed.

Received: February 7, 2025; Revised: May 23, 2025; Accepted: May 23, 2025

---

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.

---

KEY WORDS: recombinant proteins and peptides, mini-inteins, endonuclease domain, thermostability

DOI: 10.1134/S0006297925600358

Publisher’s Note. Pleiades Publishing remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

---