Browsing by Author "Lai, Yi An"

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An improved method for haptoglobin 1-1, 2-1, and 2-2 purification using monoclonal antibody affinity chromatography in the presence of sodium dodecyl sulfate
(2007-01-15)
Yueh, Sunny C. H.
;
Lai, Yi An
;
Chen, Wen Liang
;
Hsu, Hsiao Han
;
Mao, Simon J. T.
Human haptoglobin (Hp) is classified as three phenotypes: Hp 1-1, 2-1, and 2-2. Previously, we had isolated this protein by affinity columns using either hemoglobin or monoclonal antibody (mAb) prepared against Hp beta-chain (clone 8B1-3A). The isolated Hp from both methods, however, contaminates plasma apolipoprotein A-I (apoA-I). In the present report, we have developed a novel affinity column procedure using an mAb prepared against a-chain of Hp (clone 3H8) for Hp purification. Plasma was first chromatographed onto the column followed by a normal wash with a buffer containing 0.12 M NaCl and 0.02 M phosphate, pH 7.4 (PBS). The bound proteins were then prewashed with a 0.04% sodium dodecyl sulfate (SDS)-PBS, pH 7.4, to remove the low-affinity bound apoA-I from Hp. Finally, the bound Hp was eluted with a 0.1% SDS-PBS, pH 11, and collected in tubes containing 1 M Tris-HC1, pH 6.8. As a result, the isolated Hp was devoid of apoA-1 and was able to retain the biological function by forming an Hp-hemoglobin complex. The homogeneity of each isolated Hp 1-1, 2-1, or 2-2 was greater than 95% with an yield greater than 50%. The procedure described here is significantly improved in time consumption, recovery, and purity. The rationale, design, and optimization for each step are described in detail. (c) 2006 Elsevier B.V. All rights reserved.
Evidence of tandem repeat and extra thiol-groups resulted in the polymeric formation of bovine haptoglobin: A unique structure of Hp 2-2 phenotype
(2007-11-30)
Lai, Yi An
;
Lai, I. Hsiang
;
Tseng, Chi Feng
;
Lee, James
;
Mao, Simon J. T.
Human plasma Hp is classified as 1-1, 2-1, and 2-2. They are inherited from two alleles Hp 1 and Hp 2, but there is only Hp 1 in almost all the animal species. Hp 2-2 molecule is extremely large and heterogeneous associated with the development of inflammatory-related diseases. In this study, we expressed entire bovine Hp in E. coli as a alpha beta linear form. Interestingly, the antibodies prepared against this form could recognize the subunit of native Hp. In stead of a complicated column method, the antibody was able to isolate bovine Hp via immunoaffinity and gel-filtration columns. The isolated Hp is polymeric containing two major molecular forms (660 and 730 kDa). Their size and hemoglobin binding complex are significantly larger than that of human Hp 2-2. The amino-acid sequence deducted from the nucleotide sequence is similar to human Hp 2 containing a tandem repeat over the cc chain. Thus, the Hp 2 allele is not unique in human. We also found that there is one additional -SH group (Cys-97) in bovine alpha chain with a total of 8 -SH groups, which may be responsible for the overall polymeric structure that is markedly different from human Hp 2-2. The significance of the finding and its relationship to structural evolution are also discussed.