Reversible chemoenzymatic labeling of native and fusion carrier protein motifs

1. A method of forming an Apo-acyl carrier protein (ACP) from an ACP-phosphopantetheine conjugate, said method comprising:

(i) contacting an ACP-phosphopantetheine conjugate with an ACP hydrolase, wherein said ACP-phosphopantetheine conjugate comprises an phosphopantetheine analogue moiety covalently bonded to an ACP through a phosphodiester linker; and

(ii) allowing said ACP hydrolase to cleave said phosphodiester linker thereby forming an Apo-ACP,

wherein said ACP-phosphopantetheine conjugate has the formula:

wherein;

ACP is an ACP protein moiety or an ACP protein fusion moiety;

L 1 , L 2 and L 3 are independently substituted or unsubstituted alkylene;

L 4 is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene;

X is —NH— or —O—;

R 1 is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, a detectable moiety or a reactive probe.

2. The method of claim 1 , wherein said ACP is an ACP protein fusion moiety.

3. The method of claim 2 , wherein said ACP protein fusion moiety comprises an ACP protein moiety bound to an amino terminus or a carboxy terminus of a second fusion protein moiety.

4. The method of claim 2 , wherein said ACP protein fusion moiety comprises an ACP protein moiety bound to an internal amino acid residue of a second fusion protein moiety.

5. The method of claim 1 , wherein said ACP hydrolase is a P. aeruginosa ACP hydrolase, Cyanothece sp. ACP hydrolase, or P. fluorescens ACP hydrolase.

6. The method of claim 1 , wherein said apo-ACP is a Fatty Acid apo-ACP, Polyketide apo-ACP, or Peptide apo-ACP.

7. The method of claim 6 , wherein said apo-ACP is a E. coli apo-ACP, P. aeruginosa apo-ACP, S. oneidensis apo-ACP, P. falciparum apo-ACP, M. tuberculosis apo-ACP, S. coelicolor apo-ACP, A. parasiticus apo-ACP, G. fujikuroi apo-ACP, L. majuscule apo-ACP or P. fluorescens apo-ACP.

8. The method of claim 1 , further comprising:

(i) contacting said Apo-ACP with a CoA-phosphopantetheine analogue and a phosphopantetheinyl transferase, wherein said CoA-phosphopantetheine analogue comprises a phosphopantetheine analogue moiety covalently bonded to a phosphoadenosine moiety through a phosphodiester linkage;

(ii) allowing said phosphopantetheinyl transferase to cleave said phosphodiester linkage and bind said phosphopantetheine analogue moiety to said apo-ACP through a phosphodiester linker thereby forming a second ACP-phosphopantetheine conjugate;

wherein said CoA-phosphopantetheine analogue has the formula:

wherein;

L 1A , L 2A and L 3A are independently substituted or unsubstituted alkylene;

L 4A is a bond, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene;

X A is —NH— or —O—;

R 1A is substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl or substituted or unsubstituted heteroaryl, a detectable moiety or a reactive probe.

9. The method of claim 8 , further comprising

(i) contacting said second ACP-phosphopantetheine conjugate with a second ACP hydrolase; and

(ii) allowing said second ACP hydrolase to cleave said phosphodiester linker thereby forming said Apo-ACP.

10. The method of claim 8 , wherein said phosphopantetheinyl transferase is a B. subtilis phosphopantetheinyl transferase.

11. The method of claim 1 , wherein the ACP comprises-an amino acid sequence having the formula:

-DSL(Aaa1)(Aaa2)(Aaa3)(Aaa4)(Aaa5)(Aaa6)-  (I),

wherein;

Aaa1 is D, E, or S;

Aaa2 is T, F, or W,

Aaa3 is V, L, or I;

Aaa4 is E, A, or L;

Aaa5 is A, S, R, or L;

Aaa6 is V, K, or L; and

wherein said sequence is not -DSLDTVELV- (SEQ ID NO:97).

12. The method of claim 11 , wherein the ACP has the formula:

R N -DSL(Aaa1)(Aaa2)(Aaa3)(Aaa4)(Aaa5)(Aaa6)-R C

R N -DSLEFIASKLA-R C (Ia), or R N -GDSLSWLLRLLN-R C (Ib) (SEQ ID NO:2),

wherein;

R N is —NH 2 , a detectable moiety, or a reactive probe; and

R C is —COOH, a detectable moiety, a reactive probe, or -L 7 -R C1 ;

L 7 is a substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene or substituted or unsubstituted heteroarylene -L 5 -PEG-L 6 -R C1 ; wherein

L 5 and L 6 are independently —O—, —S—, —NH—, —NHC(O)—, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene; and

R C1 is a solid support comprising a cellulose membrane, a nanoparticle or a resin.

13. The method of claim 1 , wherein R 1 is a detectable moiety.

14. The method of claim 1 , wherein R 1 is a reactive probe.

15. The method of claim 1 , wherein X is —NH—.

16. The method of claim 1 , wherein X is —O—.