Cloning and expression of human haptoglobin subunits in Escherichia coli: Delineation of a major antioxidant domain

Human plasma haptoglobin (Hp) comprises α and β subunits. The α subunit is heterogeneous in size, therefore isolation of Hp and its subunits is particularly difficult. Using Escherichia coli, we show that α1, α2, β, and α2β chain was abundantly expressed and primarily present in the inclusion bodies consisting of about 30% of the cell-lysate proteins. Each cloned subunit retained its immunoreactivity as confirmed using antibodies specific to α or β chain. By circular dichroism, the structure of each expressed subunit was disordered as compared to the native Hp. The antioxidant activity was found to be associated with both α and β chains when assessed by Cu2+-induced oxidation of low density lipoprotein (LDL). Of remarkable interest, the antioxidant activity of β chain was extremely potent and markedly greater than that of native Hp (3.5×), α chain (10×) and probucol (15×). The latter is a clinically proved potent compound used for antioxidant therapy. The “unrestricted” structure of β subunit may therefore render its availability for free-radical scavenge, which provides a utility for the future design of a “mini-Hp” in antioxidant therapy. It may also provide a new insight in understanding the mechanism involved in the antioxidant nature of Hp.