title: An improved bimolecular fluorescence complementation tool based on superfolder green fluorescent protein.
abstract: Bimolecular fluorescence complementation (BiFC) has been widely used in the analysis of protein-protein interactions (PPIs) in recent years. There are many notable advantages of BiFC such as convenience and direct visualization of PPI in cells. However, BiFC has one common limitation: the separated non-fluorescent fragments can be spontaneously self-assembled into an intact protein, which leads to false-positive results. In this study, a pair of complementary fragments (sfGFPN and sfGFPC) was constructed by splitting superfolder GFP (sfGFP) between the 214 and 215 amino acid residue, and sfGFPC was mutated by site-directed gene mutagenesis to decrease the signal of negative control. Our results showed that mutations in sfGFPC (sfGFPC(m12)) can effectively decrease the signal of negative control. Thus, we provide an improved BiFC tool for the analysis of PPI. Further, since the self-assembly problem is a common shortcoming for application of BiFC, our research provides a feasible strategy for other BiFC candidate proteins with the same problem.
authors.affiliation: Key Laboratory for Biomechanics and Mechanobiology of Ministry of Education, School of Biological Science and Biomedical Engineering, Beihang University, Beijing, China.
authors.forename: Jun
authors.initials: J
authors.lastname: Zhou
authors.forename: Jian
authors.initials: J
authors.lastname: Lin
authors.forename: Cuihong
authors.initials: C
authors.lastname: Zhou
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Deng
authors.forename: Bin
authors.initials: B
authors.lastname: Xia
mesh_terms: D045744:Cell Line, Tumor; D003165:Complement System Proteins / Q000235:genetics / Q000378:metabolism; D005189:False Positive Reactions; D005816:Genetic Complementation Test / Q000295:instrumentation* / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics* / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D010446:Peptide Fragments / Q000737:chemistry*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D003165:Complement System Proteins