Split-superpositive GFP reassembly is a fast, efficient, and robust method for detecting protein-protein interactions in vivo

• Published in: Molecular bioSystems Vol. 8; no. 8; p. 2036
• Main Authors: Blakeley, Brett D, Chapman, Alex M, McNaughton, Brian R
• Format: Journal Article
• Language: English
• Published: England 01-08-2012
• Subjects: Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - metabolism; Protein Binding; Protein Structure, Secondary; Proteins - chemistry; Proteins - metabolism
• ISSN: 1742-2051
• DOI: 10.1039/c2mb25130b

Split-GFP reassembly is an operationally simple in vivo technique used to identify and study interactions involving proteins and/or peptides. However, the instability of split-GFP fragments and their susceptibility to aggregation place limitations on the broader use of split-GFP reassembly. Supercharged proteins, including supercharged GFP, are variants with high theoretical negative or positive charge that are resistant to aggregation. We show that a split-superpositive GFP (split-spGFP) variant reassembles faster and more efficiently than previously reported split-sg100 GFP and split-folding-reporter GFP (split-frGFP) systems. In addition, interaction-dependent split-spGFP reassembly is efficient at physiological temperature. The increased efficiency and robustness of split-spGFP reassembly make this reporter system ideal for identifying and studying interactions involving proteins and/or peptides in vivo, and may be particularly useful for identifying or studying interactions involving proteins or peptides that are themselves susceptible to aggregation.