title: Characterization of recombinant proteins: biochemical and biophysical criteria for the identity of native and renatured human tissue plasminogen activator and its mutants.
abstract: Rapid progress in recombinant DNA techniques has led to the production of a great variety of native and modified proteins in bacteria which, however, frequently occur in non-native conformations or in aggregated form. Renaturation methods must necessarily include criteria for the confirmation of the native structure and function of the refolded macromolecules. This Review describes some of the spectroscopic and thermodynamic techniques which can provide powerful tests for the identity of native and renatured proteins. Emphasis was laid on the concentration requirements of the methods and on the ease of application and availability of instrumentation. As an example we used the single-chain recombinant tissue plasminogen activator (rt-PA) at different pH values and buffer systems. t-PA is a monomeric serine proteinase of M(r) 68,000 that converts plasminogen into plasmin, thereby promoting the degradation of the fibrin network. This enzyme is a promising candidate for thrombolytic therapy of acute myocardial infarction. On the basis of the cDNA sequence the 527 amino acids of t-PA are likely to occur in five distinct structural domains. Specifically we exemplify the application of microcalorimetric studies for the verification of structural identity on rt-PA.
authors.affiliation: Abteilung Biophysikalische Chemie der Universität Münster, Federal Republic of Germany.
authors.forename: T
authors.initials: T
authors.lastname: Vogl
authors.forename: H J
authors.initials: HJ
authors.lastname: Hinz
mesh_terms: D000595:Amino Acid Sequence; D055592:Biophysical Phenomena; D001703:Biophysics; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D010959:Tissue Plasminogen Activator / Q000737:chemistry* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator