The modular construction of the eye lens beta gamma-crystallins makes them good candidates for protein engineering to ascertain the rules of assembly of oligomers. X-ray studies have shown that although the polypeptide chains of beta B2-crystallin and gamma-crystallins fold to form similar N- and C-terminal domains, the conformation of the connecting peptides are such that the gamma-crystallins are monomers and the beta-crystallin is a dimer. Unlike gamma-crystallins, the numerous beta-crystallins have extensions of variable sequence from the globular domains. We have tested the effect of removing the N- and C-terminal extensions from rat beta B2-crystallin using a bacterial expression system. Abundant proteins were produced in Escherichia coli using the pET or pQE vectors. Full-length and truncated proteins were purified and checked for refolding using circular dichroism. Sizing of the truncated proteins using gel filtration chromatography showed that the absence of either the N- or C-terminal extension does not affect dimerization of beta B2-crystallin.
Rapid advances in site-directed mutagenesis and total gene synthesis combined with new expression systems in prokaryotic and eukaryotic cells have provided the molecular biologist with tools for modification of existing proteins to improve catalytic activity, stability and selectivity, for construction of chimeric molecules and for synthesis of completely novel molecules that may be endowed with some useful activity. Such protein engineering can be seen as a cycle in which the structures of engineered molecules are studied by X-ray analysis and two-dimensional nuclear magnetic resonance. The results are used in the improvement of the design by using knowledge-based procedures that exploit facts, rules and observations about proteins of known three-dimensional structure.
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