ALG-2 is a newly discovered Ca²⁺-binding protein, which has been demonstrated to be directly linked to apoptosis. Structurally, ALG-2 is expressed as a single polypeptide chain corresponding to a 22 kDa protein containing five putative EF-hand Ca²⁺-binding sites. In this work, we have developed an efficient expression and purification scheme for recombinant ALG-2. Utilizing this protocol, we can routinely obtain purified recombinant protein with a yield of approximately 100 mg per liter of bacterial cell cultures. Gel filtration and chemical cross-linking experiments have shown that Ca²⁺-free ALG-2 forms a weak homodimer in solution. Biochemical and spectroscopic studies of truncated and point mutants of ALG-2 demonstrated that the fifth EF-hand Ca²⁺-binding motif is likely to participa...[ Read more ]

ALG-2 is a newly discovered Ca²⁺-binding protein, which has been demonstrated to be directly linked to apoptosis. Structurally, ALG-2 is expressed as a single polypeptide chain corresponding to a 22 kDa protein containing five putative EF-hand Ca²⁺-binding sites. In this work, we have developed an efficient expression and purification scheme for recombinant ALG-2. Utilizing this protocol, we can routinely obtain purified recombinant protein with a yield of approximately 100 mg per liter of bacterial cell cultures. Gel filtration and chemical cross-linking experiments have shown that Ca²⁺-free ALG-2 forms a weak homodimer in solution. Biochemical and spectroscopic studies of truncated and point mutants of ALG-2 demonstrated that the fifth EF-hand Ca²⁺-binding motif is likely to participate in the formation of the dimmer complex. Experimentally, both the amino- and carboxyl-terminal truncated mutants of ALG-2 have shown their ability to retain the structural, as well as, Ca²⁺-binding integrity when individually expressed in bacteria. In this respect, the N-terminal domain encompasses the first two EF-hands, and the C-terminal domain contains the remaining three EF-hands. Combining mutagenesis and spectroscopic studies, we showed that ALG-2 possesses two strong Ca²⁺-binding sites. Employing fluorescence spectroscopy and circular dichroism, we showed that the binding of Ca²⁺ to ALG-2 induced significant conformational changes in both the N-terminal and C-terminal domains of the protein. Furthermore, our studies demonstrated that Ca²⁺ binding to both strong Ca²⁺-binding sites of ALG-2 is required for ion-induced aggregation of the protein. We also report here the expression, purification, and partial characterization of a Ca²⁺-binding-deficient ALG-2 mutant (Glu47Ala/Glull4Ala). In light of its much-decreased affinity for Ca²⁺, this mutant could prove to be instrumental in elucidation the Ca²⁺-mediated function of ALG-2 within the context of its cellular environment.