Cellular depletion of biomolecular targets
View Patent ↗The present application relates to methods, compositions and systems for the specific, controllable degradation of targeted proteins. Typically, the target proteins have a function which has not yet been elucidated. The disclosure enables one to study the effect of degrading a targeted protein, which in turn, will lead to a characterization of its function. In one embodiment, the invention pertains to a composition comprising a construct wherein the construct includes a peptide including a degradation tag. The degradation tag includes a ClpX binding sequence appended to a protein, wherein the sequence is YALAA.
1. A construct for making a fusion protein, comprising:
(i) a nucleotide sequence encoding a degradation tag having a ClpX binding sequence comprising SEQ ID NO:4 for a ClpXP protease, wherein the nucleotide sequence does not encode a binding sequence for at least one of ClpA and SspB,
(ii) an antibiotic resistance marker, and
(iii) flanking sequences, wherein the flanking sequences comprise recognition sequences that correspond to sequences of a gene encoding a target protein.
2. The construct of claim 1 , wherein the nucleotide sequence encoding the degradation tag further encodes a detection marker, a purification marker, or both.
3. The construct of claim 1 , wherein the nucleotide sequence encoding the degradation tag further encodes a FLAG epitope, a His6 epitope, or a combination thereof.
4. A method of controlling degradation of a target protein, the method comprising:
expressing a fusion protein in a host cell, wherein the host cell comprises a ClpXP protease activity with respect to the expressed fusion protein, wherein the fusion protein comprises a target protein endogenous to the host cell and a degradation tag having a ClpX binding sequence comprising SEQ ID NO:4 for a ClpXP protease, and wherein the fusion protein does not bind at least one of ClpA and SspB.
5. The method of claim 4 , wherein the target protein is S1 and wherein the host cell is E. coli.
6. The method of claim 4 , wherein the fusion protein further comprises a FLAG epitope.
7. The method of claim 4 , wherein the fusion protein further comprises a His 6 epitope.
8. The method of claim 4 , wherein expressing the fusion protein comprises:
providing to the host cell a genetic construct comprising (i) a nucleotide sequence encoding the degradation tag having the ClpX binding sequence comprising SEQ ID NO:4 for the ClpXP protease and (ii) flanking sequences, wherein the flanking sequences comprise recognition sequences that correspond to sequences of the gene encoding the target protein;
inserting into an open reading frame of the gene encoding the target protein the genetic construct; and
expressing the genetic construct.
9. The method of claim 8 , wherein the flanking sequences direct recombination to the 3′ end of the gene encoding the target protein.
10. The method of claim 4 , wherein the fusion protein further comprises a linker.
11. The method of claim 4 , wherein the fusion protein further comprises an antibiotic resistance marker.
12. The method of claim 11 , wherein the antibiotic resistance marker is kanR, tetR, or ampR.
13. The method of claim 4 , wherein the fusion protein further comprises a FLAG epitope, a His 6 epitope, an antibiotic resistance marker or a combination thereof.
14. The method of claim 8 , wherein the construct further comprises nucleotide sequences for a linker.
15. The method of claim 8 , wherein the construct further comprises (iii) nucleotide sequences for an antibiotic resistance marker.
16. The method of claim 15 , wherein the antibiotic resistance marker is kanR, tetR, or ampR.
17. The method of claim 8 , wherein the construct further comprises (iii) a nucleotide sequence for a FLAG epitope.
18. The method of claim 8 , wherein the construct further comprises (iii) a nucleotide sequence for His 6 epitope.
19. The method of claim 8 , wherein the construct further comprises (iii) nucleotide sequences for a FLAG epitope, a His 6 epitope, an antibiotic resistance marker or a combination thereof.