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

O S Pavlenko, O A Gra, O N Mustafaev, K V Kabarbaeva, N S Sadovskaya, A A Tyurin, V S Fadeev, I V Goldenkova-Pavlova

Journal: Biochemistry. Biokhimiia 2019;84(8):931-940

PMID: 31522675

Full Text on Biochemistry (Moscow) View on PubMed

Abstract

Clostridium thermocellum lichenase (endo-β-1,3;1,4-glucan-D-glycosyl hydrolase, EC 3.2.1.73 (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.

Address: Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, 127276, Russia. [email protected].; Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, 127276, Russia.; Baku State University, Department of Biophysics and Molecular Biology, Baku, AZ1106, Azerbaijan. [email protected].; Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, 127276, Russia. [email protected].

DOI: 10.1134/S0006297919080091

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MeSH Terms: Bacterial Proteins, Cloning, Molecular, Clostridium thermocellum, DNA Transposable Elements, Escherichia coli, Fluorescence, Glycoside Hydrolases, Green Fluorescent Proteins, Luminescent Proteins, Microscopy, Fluorescence, Protein Domains, Protein Engineering, Protein Refolding, Recombinant Fusion Proteins, Temperature, Red Fluorescent Protein