Hetero-oligomeric tagging diminishes non-specific aggregation of target proteins fused with Anthozoa fluorescent proteins.
The tendency for tetramerization is the main disadvantage in the green fluorescent protein homologues from Anthozoa species. We report a universal method called hetero-oligomeric tagging, which diminishes troublesome consequences of tetramerization of Anthozoa-derived fluorescent proteins (FP) in intracellular protein labelling. This approach is based on the co-expression of the FP-tagged protein of interest together with an excess of free non-fluorescent FP mutant. The resulting FP heterotetramers contain only a single target polypeptide and, therefore, can be considered pseudo-monomeric. Feasibility of the method has been demonstrated with a red FP fused with cytoplasmic beta-actin or tubulin-binding protein Tau34. In addition, heterotetramers appeared to be a unique model for biophysical characterization of Anthozoa FPs in pseudo-monomeric state.
Bulina
Maria E
ME
Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry RAS, Miklukho-Maklaya 16/10, Moscow 117997, Russia.
Verkhusha
Vladislav V
VV
Staroverov
Dmitry B
DB
Chudakov
Dmitry M
DM
Lukyanov
Konstantin A
KA
D000199:Actins / Q000235:genetics / Q000378:metabolism; D000818:Animals; D037421:Anthozoa / Q000737:chemistry*; D001671:Biochemistry / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D005786:Gene Expression Regulation; D005796:Genes; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000097573:Red Fluorescent Protein
D000199:Actins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008869:Microtubule-Associated Proteins; D011993:Recombinant Fusion Proteins; D000097573:Red Fluorescent Protein