Designing a thermostable mini-intein for intein-mediated purification of recombinant proteins and peptides

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Abstract

This paper reports 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 chosen as the basis for further design of a self-cleaving carrier of affinity tags through single-point mutagenesis. We performed the C1A, D405G and C1A/D405G mutations, which were proposed to eliminate the intein’s capability of 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 at its temperature optimum around 60°C. Thus, we constructed thermostable temperature-activated mini-inteins capable of efficient protein splicing or cleavage of C-terminal extein. The engineered TthDnaE1 Δ280 C1A/D405G mini-intein can serve as basis for the development of new expression system for intein-mediated production of pharmaceutical recombinant proteins and peptides.

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About the authors

A. A. Karanov

Institute of Bioorganic Chemistry, Russian Academy of Sciences; Lomonosov Moscow State University

Author for correspondence.
Email: andrey-karanov2000@mail.ru

Faculty of Bioengineering and Bioinformatics

Russian Federation, 117997 Moscow; 119234 Moscow

E. A. Zayats

Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: refolding@mail.ru
Russian Federation, 117997 Moscow

M. A. Kostromina

Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: refolding@mail.ru
Russian Federation, 117997 Moscow

Yu. A. Abramchik

Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: refolding@mail.ru
Russian Federation, 117997 Moscow

A. R. Sharafutdinova

Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: refolding@mail.ru
Russian Federation, 117997 Moscow

M. S. Surkova

Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: refolding@mail.ru
Russian Federation, 117997 Moscow

A. A. Zamyatnin

Lomonosov Moscow State University

Email: refolding@mail.ru

Faculty of Bioengineering and Bioinformatics

Russian Federation, 119234 Moscow

R. S. Esipov

Institute of Bioorganic Chemistry, Russian Academy of Sciences; Lomonosov Moscow State University

Email: refolding@mail.ru

Faculty of Bioengineering and Bioinformatics

Russian Federation, 117997 Moscow; 119234 Moscow

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Supplementary files

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2. Fig. 1. Comparison of the structures of TthDnaE1 and the artificial mini-intein SspDnaB. The N- and C-terminal regions of the polypeptide chain that form the HINT domain are shown in green and orange, respectively. The β-strands formed by amino acid residues G87–V92 and L388–L406 are shown in blue and red, respectively.

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3. Fig. 2. Models of the structure of mini-inteins TthDnaE1 Δ272 (a), Δ280 (b) and Δ287 (c)

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4. Fig. 3. Electrophoretic analysis of splicing products of hybrid proteins containing the TthDnaE1 mini-intein with deletions Δ272, Δ280 and Δ287 (15% SDS-PAGE). Legend: “EIE” – undigested mini-intein, “EI” – residual protein without C-terminal extein, “IE” – residual protein without N-terminal extein, “I” – mini-intein without exteins, “EE” – extein ligation product

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5. Fig. 4. Electrophoretic analysis of the cleavage products of the hybrid proteins based on the TthDnaE1 Δ280 mini-intein without mutations and with point substitutions C1A, D405G and C1A/D405G. a – 15% SDS-PAGE; b – 10% tricine-SDS-PAGE. Legend – as in Fig. 3; “EN” – N-terminal extein, “EC” – C-terminal extein.

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6. Fig. 5. Cleavage products of hybrid proteins based on the mini-intein TthDnaE1 Δ280 (a, b) and its mutant forms C1A (c, d), D405G (e, f) and C1A/D405G (g, h) depending on the incubation conditions. a, c, d, g – Content of products at pH 6.0 and temperature in the range of 20–80 °C; b, d, f, h – at 60 °C and pH in the range of 6.0–9.0. The initial hybrid protein is shown in white on the diagrams, product “I” is shown in light gray, and product “EI” is shown in dark gray. The data were obtained by densitometric method for 15% SDS-PAA gels. “K” – hybrid protein before incubation

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