title: Expression and aggregation of recombinant alpha A-crystallin and its two domains.
abstract: The 20 kDa alpha A and alpha B subunits of alpha-crystallin from mammalian eye lenses form large aggregates with an average molecular weight of 800,000. To get insight into the interactions responsible for aggregate formation, we expressed in Escherichia coli the putative N- and C-terminal domains of alpha A-crystallin, as well as the intact alpha A-crystallin chain. The proteins are expressed in a stable form and in relatively high amounts (20-60% of total protein). Recombinant alpha A-crystallin and the C-terminal domain are expressed in a water-soluble form. Recombinant alpha A-crystallin forms aggregates comparable with alpha-crystallin aggregates from calf lenses, whereas the C-terminal domain forms dimers or tetramers. The N-terminal domain is expressed in an initially water-insoluble form. After solubilization, denaturation and reaggregation the N-terminal domain exists in a high molecular weight multimeric form. These observations suggest that the interactions leading to aggregation of alpha A-crystallin subunits are mainly located in the N-terminal half of the chain.
authors.affiliation: Department of Biochemistry, University of Nijmegen, Netherlands.
authors.forename: K B
authors.initials: KB
authors.lastname: Merck
authors.forename: W A
authors.initials: WA
authors.lastname: De Haard-Hoekman
authors.forename: B B
authors.initials: BB
authors.lastname: Oude Essink
authors.forename: H
authors.initials: H
authors.lastname: Bloemendal
authors.forename: W W
authors.initials: WW
authors.lastname: De Jong
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D003459:Crystallins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000235:genetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics
chemical_list: D003459:Crystallins; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011994:Recombinant Proteins