Human Carnitine Acetyl Transferase (hCAT) reversibly catalyzes the transfer of the acetyl-moiety from acetyl-CoA to L-carnitine, modulating the acetyl-CoA/CoA ratio in mitochondria. Derangement of acetyl-CoA/CoA ratio leads to metabolic alterations that could result in the onset or worsening of pathological states. Due to the importance of CAT as a pharmacological target and to the European directive for reducing animal experimentation, we have pointed out a procedure to produce a recombinant, pure, and functional hCAT using the E. coli expression system. The cDNA encoding for the hCAT was cloned into the pH6EX3 vector. This construct was used to transform the E. coli Rosetta strain. The optimal conditions for the overexpression of the fully active hCAT include induction with a low concentration of IPTG (0.01 mM) and a low growth temperature (25 degrees C). The recombinant protein was purified from bacterial homogenate by affinity chromatography. The pure hCAT is very stable in an aqueous solution, retaining full activity for at least two months if stored at - 20 degrees C. These results could be helpful for a broad set of functional studies on hCAT, including drug-design applications.

Overproduction, Purification, and Stability of the Functionally Active Human Carnitine Acetyl Transferase

Giudice D.;Console L.;Indiveri C.
2022-01-01

Abstract

Human Carnitine Acetyl Transferase (hCAT) reversibly catalyzes the transfer of the acetyl-moiety from acetyl-CoA to L-carnitine, modulating the acetyl-CoA/CoA ratio in mitochondria. Derangement of acetyl-CoA/CoA ratio leads to metabolic alterations that could result in the onset or worsening of pathological states. Due to the importance of CAT as a pharmacological target and to the European directive for reducing animal experimentation, we have pointed out a procedure to produce a recombinant, pure, and functional hCAT using the E. coli expression system. The cDNA encoding for the hCAT was cloned into the pH6EX3 vector. This construct was used to transform the E. coli Rosetta strain. The optimal conditions for the overexpression of the fully active hCAT include induction with a low concentration of IPTG (0.01 mM) and a low growth temperature (25 degrees C). The recombinant protein was purified from bacterial homogenate by affinity chromatography. The pure hCAT is very stable in an aqueous solution, retaining full activity for at least two months if stored at - 20 degrees C. These results could be helpful for a broad set of functional studies on hCAT, including drug-design applications.
Carnitine
Drug design
Metabolism
Mitochondria
Transferase enzyme
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/20.500.11770/343169
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