A physical-chemical and immunological comparison shows that native and renatured Escherichia coli tryptophan synthase beta 2 subunits are identical.
An acid-denaturation of the beta 2 subunit of Escherichia coli tryptophan synthase has been recently described. In the present study, renaturation yield of acid-denaturated beta 2, and the influence of temperature, protein concentration and presence of ligands are investigated. It is also demonstrated that 3 forms of the protein are obtained at the end of the renaturation process: one is fully active, and is identical to native beta 2, as indicated by some of its chemical and physical properties, as well as by its immunological reactivity towards monoclonal antibodies specific for the native protein. A second form is composed of high molecular weight insoluble and inactive aggregates. A third form consists of low molecular weight soluble and inactive aggregates. The results obtained for the immunochemical reactivity of these small aggregates indicate that they are formed with partly correctly folded beta monomers assembled by specific but incorrect quaternary interactions. The capacity of monoclonal antibodies to detect such incorrect structures and to characterize renatured proteins is particularly emphasized.
Murry-Brelier
A
A
Département de Biochimie et Génétique Moléculaire, Institut Pasteur, Paris, France.
Goldberg
M E
ME
D000911:Antibodies, Monoclonal; D002853:Chromatography, Liquid / Q000379:methods; D004795:Enzyme Stability; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000201:enzymology*; D007700:Kinetics; D008024:Ligands; D010446:Peptide Fragments; D011487:Protein Conformation; D011489:Protein Denaturation; D012995:Solubility; D013696:Temperature; D014367:Tryptophan Synthase / Q000032:analysis*
D000911:Antibodies, Monoclonal; D008024:Ligands; D010446:Peptide Fragments; D014367:Tryptophan Synthase