Lanthanide-binding tags as versatile protein coexpression probes.
Comprehensive proteomic analyses require new methodologies to accelerate the correlation of gene sequence with protein function. Key tools for such efforts include biophysical probes that integrate into the covalent architecture of proteins. Lanthanide-binding tags (LBTs) are expressible, multitasking fusion partners that are optimized to bind lanthanide ions and have several desirable attributes, which include long-lived luminescence, excellent X-ray scattering power for phase determination, and magnetic properties to facilitate NMR spectroscopic structure elucidation. Herein, we present peptide sequences with a 40-fold higher affinity for Tb(3+) ions and significantly brighter luminescence intensity compared with existing peptides. Incorporation of an LBT onto ubiquitin as a prototype fusion protein allows the use of powerful protein-visualization techniques, which include rapid luminescence detection of LBT-tagged proteins in SDS-PAGE gels, as well as determination of protein concentrations in complex mixtures. The LBT strategy is a new alternative for expressing fluorescent fusion proteins by routine molecular biological techniques.
Franz
Katherine J
KJ
Department of Chemistry Massachusetts Institute of Technology Cambridge, MA 02139, USA.
Nitz
Mark
M
Imperiali
Barbara
B
D000595:Amino Acid Sequence; D001665:Binding Sites; D020650:Combinatorial Chemistry Techniques; D004591:Electrophoresis, Polyacrylamide Gel; D005782:Gels; D008163:Luminescent Measurements; D015335:Molecular Probes / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D010455:Peptides / Q000138:chemical synthesis* / Q000737:chemistry / Q000235:genetics; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000138:chemical synthesis / Q000737:chemistry; D013725:Terbium / Q000737:chemistry*; D025801:Ubiquitin / Q000737:chemistry / Q000235:genetics
D005782:Gels; D015335:Molecular Probes; D010455:Peptides; D011993:Recombinant Fusion Proteins; D025801:Ubiquitin; D013725:Terbium