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).

Inteins in Science: Evolution to Application

“…The engineering of reporter proteins was done by Kawase et al by constructing engineered Npu DnaE split intein variants, where the N-intein sequence was modified by inducing Gly4Tyr and Asp5Glu mutations and the C-inteins variants were selected from libraries created by error-prone PCR. Active variants were screened by using a GFP-intein conjugates, which were used to construct a turn-on system for enzymes like human immunodeficiency 1 protease and NanoLuc luciferase [ 148 ].…”

Luminescence detection of peptide:N-glycanase activity using engineered split inteins

“…1). 16 An active variant of IntC, termed IntCmut3, showed good PTS activity with an N-intein variant (IntN; CLSGDTEILTA). A 16-residue peptide (NLs-IntN), consisting of a five-residue peptide derived from NanoLuc luciferase and IntN, can react with IntCm-NLlg that contains IntCmut3 and a NanoLuc fragment to generate an active NanoLuc.…”

“…A 16-residue peptide (NLs-IntN), consisting of a five-residue peptide derived from NanoLuc luciferase and IntN, can react with IntCm-NLlg that contains IntCmut3 and a NanoLuc fragment to generate an active NanoLuc. 16 The NanoLuc luciferase is capable of emitting very bright light, allowing sensitive detection of the PTS reaction by the luciferase assay.…”

“…1B). 16 The incorporation of GlcNAc 2 therefore reduces the affinity between IntN and IntCmut3. Peptide 3GN2 contains Asn(GlcNAc 2 ) in place of Ser at position 3 and Ser in place of Asp at position 5 in IntN.…”