Gain of structure and IgE epitopes by eukaryotic expression of the major Timothy grass pollen allergen, Phl p 1.

Approximately 400 million allergic patients are sensitized against group 1 grass pollen allergens, a family of highly cross-reactive allergens present in all grass species. We report the eukaryotic expression of the group 1 allergen from Timothy grass, Phl p 1, in baculovirus-infected insect cells. Domain elucidation by limited proteolysis and mass spectrometry of the purified recombinant glycoprotein indicates that the C-terminal 40% of Phl p 1, a major IgE-reactive segment, represents a stable domain.

Solubilizing buried domains of proteins: a self-assembling interface domain from glutathione reductase.

In the dimeric glutathione reductase (GR) from Escherichia coli, the interface domain is largely surrounded by the other three domains in each subunit of the protein. Subgenes encoding three forms of the interface domain have been expressed in E. coli and the products purified from inclusion bodies: INT is the excised interface domain, as it is found in native GR; INTN and INTFN are variants carrying exchanges of surface residues in what would have been hydrophobic contact regions with other neighboring domains.

Coenzyme binding of a folding intermediate of aspartate aminotransferase detected by HPLC fluorescence measurements.

Equilibrium dissociation and unfolding of dimeric aspartate aminotransferase from Escherichia coli proceeds via two compact monomeric intermediates which have similar hydrodynamic volumes but different fluorescence properties. We probed binding of the coenzyme pyridoxal 5'-phosphate to these intermediates by coupling fluorescence detection to size-exclusion HPLC. This procedure gave additionally an internal conformational probe of the unfolding transitions of the enzyme.