Abstract
The M90V mutant of guinea pig alpha-lactalbumin (gpLA) was expressed intracellularly in Escherichia coli using a gpLA gene fusion to the IgG-binding ('Z') domain coding sequences of staphylococcal protein A. The fusion protein was expressed as an inclusion body, then purified and refolded in vitro; CNBr cleavage of the fusion polypeptide yielded native alpha-lactalbumin. The recombinant M90V gpLA was virtually identical with natural gpLA with respect to its ability to stimulate lactose synthesis by galactosyl transferase and the recombinant and natural molecules also exhibited similar circular dichroism spectra, thermal melting profiles and NMR spectra. However, modest perturbations in the chemical shifts of amide protons in the C-helix residues, attributable to the Met-->Val mutation, were observed. In defined media, this expression system enabled the production of highly-enriched 15N- and 13C, 15N-labeled gpLA. Use of this material will allow the solution conformations of the native and molten globule states of gpLA to be characterized by high-resolution multidimensional NMR.
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