Id protein-firefly luciferase N-fragment & firefly luciferase C-fragment-MyoD protein

Firefly luciferase (Fluc) is an oxygenase extracted from with a molecular weight of 62 kDa (1). In the presence of adenosine triphosphate (ATP) and O, Fluc oxidizes the heterocyclic substrate d-luciferin to oxyluciferin and emits light in the wavelength range of 400–620 nm (2). The active site of Fluc comprises two distinct domains, a large N-terminal domain (residue 4–436) and a small C-terminal domain (residue 440–544), which are separated by a wide cleft (1). Splitting Fluc into N- and C-terminal fragments destroys its enzymatic activity, resulting in a complete loss of bioluminescence. The enzymatic activity or bioluminescence can be restored if the N- and C-terminal fragments are in close proximity (3). This led to the development of a novel labeling strategy for imaging protein–protein interactions , the split reporter (4). In this method, reporters like Fluc are dissected into two fragments and fused to a pair of proteins (A and B) that strongly interact with each other. The enzymatic activity of Fluc can be restored two split reporter approaches: a complementation strategy and a reconstitution strategy. In the complementation strategy, protein A is connected with the N-terminal fragment of Fluc, and protein B is connected with the C-terminal fragment of Fluc. Interaction between protein A and B recovers the enzymatic activity of Fluc by bringing the two fragments of Fluc closely together. In the reconstitution strategy, protein A is connected with the N-terminal of one-half of a protein splicing system such as DNA polymerase III (N-intein DnaE) followed by N-terminal fragment of Fluc. Protein B is linked to the C-terminal of the other half of the splicing system (C-intein DnaE) followed by the C-terminal fragment of Fluc. The interaction between protein A and B brings the N- and C-inteins together, resulting in the joining of N- and C-terminal fragments of Fluc by a peptide bond and a release of a fully reconstituted protein Fluc. Both split enzyme approaches allow for recovery of bioluminescence. MyoD is a myogenic regulatory protein that belongs to the basic helix-loop-helix (bHLH) family of transcription factors (5). MyoD activates myogenesis by binding directly to the control region of muscle-specific genes, and it converts fibroblasts into skeletal muscle. The helices in MyoD bind tightly with other HLH factors such as Id to form a heterodimer. Id, also known as an inhibitor of differentiation or inhibitor of DNA binding, lacks the basic domain for DNA binding and acts as dominant negative regulator in myogenesis or other cell proliferations (6). Id protein-firefly luciferase N-fragment & firefly luciferase C-fragment-MyoD protein (Id-NFluc & CFluc-MyoD) make up a pair of interacting proteins attached to the split Fluc fragments (7). Id-NFluc & CFluc-MyoD can be prepared with complementation strategy or reconstitution strategy, and are employed for imaging of the interaction between Id and MyoD the produced bioluminescence.