Activation of Protease and Luciferase Using Engineered Nostoc punctiforme PCC73102 DnaE Intein with Altered Split Position.

Abstract

Inteins, self-catalytic enzymes, have been widely used in the field of protein engineering and chemical biology. Here, Nostoc punctiforme PCC73102 (Npu) DnaE intein was engineered to have an altered split position. An 11-residue N-intein of DnaE in which Gly and Asp were substituted for Tyr4 and Glu5, respectively, was designed, and the active C-intein variants were acquired by a GFP fluorescence-based screening. The designed N-intein and the obtained active C-intein variants were used to construct a turn-on system for enzyme activities such as human immunodeficiency 1 protease and NanoLuc luciferase. Based on the NanoLuc-intein fusion, we developed two intein pairs, each of which is capable of reacting preferentially, by interchanging the charged amino acids on N- and C-inteins. The specific splicing reactions were easily monitored and discriminated by bioluminescence resonance energy transfer (BRET).

• Authors: Misaki Kawase, Meiko Fujioka, Tsuyoshi Takahashi
• Journal: Chembiochem : a European journal of chemical biology (Chembiochem) Vol. 22 Issue 3 Pg. 577-584 (02 02 2021) ISSN: 1439-7633 [Electronic] Germany
• PMID: 32969142 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: © 2020 Wiley-VCH GmbH.

Chemical References

• Luciferases
• DNA polymerase III, alpha subunit
• DNA Polymerase III
• Peptide Hydrolases

Topics

• Biocatalysis
• DNA Polymerase III (genetics, metabolism)
• Inteins
• Luciferases (metabolism)
• Nostoc (enzymology)
• Peptide Hydrolases (metabolism)
• Protein Engineering