Methods for making and using molecular switches involving circular permutation

• US 8,338,138 B2
• Filed: 01/28/2005
• Issued: 12/25/2012
• Est. Priority Date: 01/28/2004
• Status: Active Grant

• Current Assignee: Johns Hopkins University
• Original Assignee: Johns Hopkins University
• Inventors: Ostermeier, Marc A., Guntas, Gurkan
• Primary Examiner(s): Ketter, Jim
• Application Number:
  US10/588,114
• Publication Number:
  US 20090005266A1
• Time in Patent Office: 2,888 Days
• Field of Search: None
• US Class Current: 435/91.41
• CPC Class Codes: C07K 14/001 by chemical synthesis
• CPC Class Codes: C12N 15/1093 General methods of preparing gene libraries, not provided for in other subgroups
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 15/65 using markers enzymes used as markers C12N15/52
• CPC Class Codes: C12N 15/66 General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease

Abstract

The invention provides molecular switches which couple external signals to functionality, and combinatorial methods of making and using the same involving circular permutation of nucleic acid and amino acid sequences. The switches according to the invention can be used, for example, to regulate gene transcription, target drug delivery to specific cells, transport drugs intracellularly, control drug release, provide conditionally active proteins, perform metabolic engineering, and modulate cell signaling pathways. Libraries comprising the switches, expression vectors and host cells for expressing the switches are also provided.

Claims

1. A method for assembling a modulatable fusion molecule polypeptide, comprising:

generating a circular permutation of an insertion nucleic acid sequence, wherein the insertion nucleic acid sequence encodes a polypeptide that recognizes an input signal; and

inserting the insertion sequence into an acceptor nucleic acid sequence, wherein the acceptor sequence encodes a polypeptide that produces an output signal provided the output signal is not fluorescence, wherein the fused insertion and acceptor sequences encode a modulatable fusion polypeptide having the output signal functionally coupled to the input signal.

2. The method according to claim 1, wherein the insertion sequence is inserted at a selected site in the acceptor sequence.

3. The method according to claim 1, wherein the insertion sequence is inserted at a random site in the acceptor sequence.

4. A method for assembling a modulatable fusion molecule polypeptide, comprising:

generating a circular permutation of an insertion nucleic acid sequence, wherein the insertion nucleic acid sequence encodes a polypeptide that produces an output signal provided the output signal is not fluorescence; and

inserting the insertion sequence into an acceptor nucleic acid sequence, wherein the acceptor sequence encodes a polypeptide that recognizes an input signal, wherein the fused insertion and acceptor sequences encode a modulatable fusion polypeptide having the output signal functionally coupled to the input signal.

5. The method according to claim 4, wherein the insertion sequence is inserted at a selected site in the acceptor sequence.

6. The method according to claim 4, wherein the insertion sequence is inserted at a random site in the acceptor sequence.

7. A method for assembling a modulatable fusion molecule polypeptide, comprising:

generating a circular permutation of an acceptor nucleic acid sequence, wherein the acceptor nucleic acid sequence encodes a polypeptide that recognizes an input signal; and

inserting the insertion sequence into an acceptor nucleic acid sequence, wherein the insertion sequence encodes a polypeptide that produces an output signal provided the output signal is not fluorescence, wherein the fused insertion and acceptor sequences encode a modulatable fusion polypeptide having the output signal functionally coupled to the input signal.

8. The method according to claim 7, wherein the insertion sequence is inserted at a selected site in the acceptor sequence.

9. The method according to claim 7, wherein the insertion sequence is inserted at a random site in the acceptor sequence.

10. A method of assembling a modulatable fusion molecule polypeptide, comprising:

generating a circular permutation of an acceptor nucleic acid sequence, wherein the acceptor nucleic acid sequence encodes a polypeptide that produces an output signal provided the output signal is not fluorescence; and

inserting the insertion sequence into an acceptor nucleic acid sequence, wherein the insertion sequence encodes a polypeptide that recognizes an input signal, wherein the fused insertion and acceptor sequences encode a modulatable fusion polypeptide having the output signal functionally coupled to the input signal.

11. The method according to claim 10, wherein the insertion sequence is inserted at a selected site in the acceptor sequence.

12. The method according to claim 10, wherein the insertion sequence is inserted at a random site in the acceptor sequence.