Biochemistry (Moscow)

, Volume 84, Issue 8, pp 931–940 | Cite as

Thermostable Lichenase from Clostridium thermocellum as a Host Protein in the Domain Insertion Approach

  • O. S. PavlenkoEmail author
  • O. A. Gra
  • O. N. MustafaevEmail author
  • K. V. Kabarbaeva
  • N. S. Sadovskaya
  • A. A. Tyurin
  • V. S. Fadeev
  • I. V. Goldenkova-PavlovaEmail author


Clostridium thermocellum lichenase (endo-β-1,3;1,4-glucan-D-glycosyl hydrolase, EC (P29716)) has been tested for the insertion of two model fluorescent proteins (EGFP and TagRFP) into two regions of this enzyme. Functional folding of the resulting proteins was confirmed by retention of lichenase activity and EGFP and TagRFP fluorescence. These results convincingly demonstrate that (i) the two experimentally selected lichenase loop regions may serve as the areas for domain insertion without disturbing enzyme folding in vivo; (ii) lichenase permits not only single but also tandem insertions of large protein domains. High specific activity, outstanding thermostability, and efficient in vitro refolding of thermostable lichenase make it an attractive new host protein for the insertional fusion of domains in the engineering of multifunctional proteins.


lichenase fluorescent proteins single and tandem insertions of proteins 





phosphoglycerate kinase


total soluble protein


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Funding. This work was supported by the Russian Foundation for Basic Research (project 16-34-00249; AAT).

Conflict of interest. The authors declare no conflict of interest in financial or any other sphere.

Ethical approval. This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10541_2019_809_MOESM1_ESM.pdf (187 kb)


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Copyright information

© Pleiades Publishing, Ltd. 2019

Authors and Affiliations

  1. 1.Timiryazev Institute of Plant PhysiologyRussian Academy of SciencesMoscowRussia
  2. 2.Baku State University, Department of Biophysics and Molecular BiologyBakuAzerbaijan

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