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. 1995 Oct 24;92(22):10147–10151. doi: 10.1073/pnas.92.22.10147

Mutagenesis of the human apolipoprotein B gene in a yeast artificial chromosome reveals the site of attachment for apolipoprotein(a).

S P McCormick 1, J K Ng 1, S Taylor 1, L M Flynn 1, R E Hammer 1, S G Young 1
PMCID: PMC40753  PMID: 7479743

Abstract

Lipoprotein(a) [Lp(a)] is a lipoprotein formed by the disulfide linkage of apolipoprotein (apo) B100 of a low density lipoprotein particle to apolipoprotein(a). Prior studies have suggested that one of the C-terminal Cys residues of apo-B100 is involved in the disulfide linkage of apo-B100 to apo(a). To identify the apo-B100 Cys residue involved in the formation of Lp(a), we constructed a yeast artificial chromosome (YAC) spanning the human apo-B gene and used gene-targeting techniques to change Cys-4326 to Gly. The mutated YAC DNA was used to generate transgenic mice expressing the mutant human apo-B100 (Cys4326Gly). Unlike the wild-type human apo-B100, the mutant human apo-B100 completely lacked the ability to bind to apo(a) and form Lp(a). This study demonstrates that apo-B100 Cys-4326 is required for the assembly of Lp(a) and shows that gene targeting in YACs, followed by the generation of transgenic mice, is a useful approach for analyzing the structure of large proteins coded for by large genes.

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