title: Correctly folded T-cell receptor fragments in the periplasm of Escherichia coli. Influence of folding catalysts.
abstract: The T-cell receptor is the central recognition molecule in cellular immunity. Its extracellular domains are homologous with and thought to be structurally similar to an antibody Fab fragment. Despite the biological importance of the TCR and the ease of bacterial expression of antibody fragments, there are only few reports of TCR-fragment expression in E. coli. In order to understand the difficulties of expressing correctly folded TCR fragments in E. coli, we have characterized the expression behavior of single-chain Fv analogs of three different TCRs (scTCR). All of them can be folded into the correct conformation in the periplasm of E. coli, yet the extent of correct folding varies greatly. In order to overcome the folding problems of some of the scTCRs, we have developed a system with enhanced in vivo folding capability based on the simultaneous induction of the heat-shock response and over-expression of the E. coli disulfide isomerase DsbA at low temperature. We present a model describing the folding of the scTCRs in the periplasm of E. coli and possible points of folding assistance. The role of the periplasm as an independent folding compartment is emphasized and the existence of a general periplasmic chaperone is postulated. We have also shown that a bivalent scTCR, dimerized in vivo with helix-turn-helix modules, can be expressed in a correctly folded form.
authors.affiliation: Max-Planck-Institut für Biochemie, Protein Engineering Group, Martinsried, Germany.
authors.forename: C
authors.initials: C
authors.lastname: Wülfing
authors.forename: A
authors.initials: A
authors.lastname: Plückthun
mesh_terms: D001483:Base Sequence; D004926:Escherichia coli / Q000378:metabolism*; D018448:Models, Immunological; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D017510:Protein Folding; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D012995:Solubility; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011948:Receptors, Antigen, T-Cell; D011994:Recombinant Proteins