Cloning and expression of human haptoglobin subunits in Escherichia coli: delineation of a major antioxidant domain.
Human plasma haptoglobin (Hp) comprises alpha and beta subunits. The alpha subunit is heterogeneous in size, therefore isolation of Hp and its subunits is particularly difficult. Using Escherichia coli, we show that alpha1, alpha2, beta, and alpha2beta 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 alpha or beta 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 alpha and beta chains when assessed by Cu(2+)-induced oxidation of low density lipoprotein (LDL). Of remarkable interest, the antioxidant activity of beta chain was extremely potent and markedly greater than that of native Hp (3.5x), alpha chain (10x) and probucol (15x). The latter is a clinically proved potent compound used for antioxidant therapy. The "unrestricted" structure of beta 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.
Lai
I Hsiang
IH
Research Institute of Biochemical Engineering, College of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan, ROC.
Tsai
Tsung I
TI
Lin
Hong Huei
HH
Lai
Wei Yen
WY
Mao
Simon J T
SJ
D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression / Q000502:physiology*; D006242:Haptoglobins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D000975:Antioxidants; D006242:Haptoglobins; D021122:Protein Subunits; D011994:Recombinant Proteins