Bis-biotinylation tags

• US 9,678,080 B2
• Filed: 06/12/2014
• Issued: 06/13/2017
• Est. Priority Date: 06/14/2013
• Status: Active Grant

• Current Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Original Assignee: Pacific Bioscience of California Incorporated (L'Oreal SA)
• Inventors: Miller, Erik, Kamtekar, Satwik, Bjornson, Keith, Hanes, Jeremiah
• Primary Examiner(s): Weiler, Karen S
• Application Number:
  US14/303,296
• Publication Number:
  US 20150011433A1
• Time in Patent Office: 1,097 Days
• Field of Search: None
• CPC Class Codes: C07K 14/36 from Actinomyces; from Streptomyces (G)
• CPC Class Codes: G01N 33/58 involving labelled substances G01N33/53 takes precedence

Abstract

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.

Claims

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.