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Expression and Purification of ATP-dependent Human Lon Protease and Characterization of its Enzymatic Activity
Yao Wei1,4#, Xia Lei1,2,3#, Liu Yongzhang1,3,4#, Li Zhen1,3,4, Wang Lu1,2,3, Lü Bin1,2,3,4*
1Institute of Biophysics, Wenzhou Medical College, Wenzhou 325035, China; 2Attardi Institute of Mitochondrial Biomedicine, Wenzhou Medical College, Wenzhou 325035, China; 3Zhejiang Key Laboratory of Medical Genetics, Wenzh
Abstract: Human Lon protease is a homo-oligomeric ring-shaped ATP-dependent protease located in mitochondrial matrix. Recent researches demonstrated that Lon protease plays an important role in cellular homeostasis， mitochondrial protein quality control and metabolic regulation. Human Lon expression plasmid carrying a carboxyl-terminal hexahistidine tag was transformed into the E.coli Rosetta 2 strain and we optimized the induction condition for high expression. Ni2+-NTA agarose was used to purify this his-tag Lon protease. The peptidase activity of Lon was measured by using peptide substrate Rhodamine 110， bis-(CBZ-L-alanyl-L-alanine amide)[(Z-AA)2 Rh110]， Casein and purified TFAM as substrates. The results showed that purified Lon retained the ATP-dependent enzymatic activity.
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