Expression and aggregation of recombinant αA-crystallin and its two domains

• Published in: Biochimica et biophysica acta. Gene structure and expression Vol. 1130; no. 3; pp. 267 - 276
• Main Authors: Merck, Karin B., De Haard-Hoekman, Willeke A., Oude Essink, Belinda B., Bloemendal, Hans, De Jong, Wilfried W.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 06.04.1992; Elsevier
• Subjects: Aggregate formation; Biological and medical sciences; Fundamental and applied biological sciences. Psychology; Gene expression; Heat shock protein; Molecular and cellular biology; Molecular genetics; Protein domain; Protein engineering; α-Crystallin; Protein engineering; CBB; SDS; HSP; Aggregate formation; α-Crystallin; PMSC; USF; PAGE; WSF; αA2D; αA1D; bp; ds; PAA; UIF; HMW; Protein domain; Heat shock protein; LMW; IPTG; Heterologous system; Escherichia coli; Recombinant DNA; Gene expression; Aggregation; Eye; Proteins; Vertebrata; Crystallin; Mammalia; Bacteria; Alpha-Peptide chain; Enterobacteriaceae
• ISSN: 0167-4781; 1879-2634
• DOI: 10.1016/0167-4781(92)90439-7

The 20 kDa αA and αB subunits of α-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 αA-crystallin, as well as the intact αA-crystallin chain. The proteins are expressed in a stable form and in relatively high amounts (20–60% of total protein). Recombinant αA-crystallin and the C-terminal domain are expressed in a water-soluble form. Recombinant αA-crystallin forms aggregates comparable with α-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 αA-crystallin subunits are mainly located in the N-terminal half of the chain.