Bis-biotinylation tags
Multi-biotinylated reactants are provided which can be used in divalent complexes for various applications such as colocalization, labeling, immobilization, and purification. Methods for constructing, purifying, and using the bis-biotinylated reactants are also provided. In certain embodiments, two bis-biotinylated reactants are bound to a single streptavidin tetramer to provide a complex having a 1:1 stoichiometry with respect to the bis-biotinylated reactants.
1. A composition comprising a protein comprising two biotin ligase recognition sequences oriented in tandem at an N-terminal or C-terminal end of the protein, each biotin ligase recognition sequence having a biotin moiety covalently linked thereto, wherein the two biotin ligase recognition sequences and the two biotins collectively constitute a first bis-biotin tag; a solid support covalently coupled to a second bis-biotin tag; and a tetravalent biotin-binding agent to which both bis-biotin tags are bound.
2. The composition of claim 1 , wherein the protein is a polymerase.
3. The composition of claim 1 , wherein the two biotin moieties in the first bis-biotin tag are separated by 20-60 angstroms.
4. The composition of claim 1 , wherein the tetravalent biotin-binding agent is a streptavidin tetramer.
5. The composition of claim 4 , wherein the streptavidin tetramer comprises a first streptavidin dimer bound to both biotin moieties of the first bis-biotin tag, and a second streptavidin dimer bound to both biotin moieties of the second bis-biotin tag.
6. A method for immobilizing a protein, the method comprising:
a) providing a protein comprising two biotin ligase recognition sequences oriented in tandem at an N-terminal or C-terminal end of the protein, each biotin ligase recognition sequence having a biotin moiety covalently linked thereto, wherein the two biotin ligase recognition sequences and the two biotins collectively constitute a first bis-biotin tag;
b) covalently linking a solid support to a second bis-biotin tag;
c) binding the protein to a tetravalent biotin-binding agent, thereby producing a first complex comprising the protein bound to the tetravalent biotin-binding agent; and
d) exposing the solid support bearing the second bis-biotin tag to the first complex, thereby immobilizing the protein on the solid support by producing a second complex comprising the solid support bound to the first complex.
7. The method of claim 6 , further comprising isolating the first complex prior to said exposing the solid support.
8. The method of claim 6 , further comprising isolating the second complex.