title: A method for directly determining intact recombinant insulin fusion proteins in crude fermentation extracts.
abstract: In evaluating the purification of genetically engineered human insulin there is no plausible correlation between the yield of expression as determined by SDS-PAGE (taking into account all normally occurring losses during the several purification steps) and the actual yield, i.e., the final pure product. The aim of our work was to develop a fast, accurate, and reproducible method for the quantification of the initial yield of the intact insulin fusion protein directly after fermentation and without prior purification of the fermentation product. Therefore, a protocol for efficient tryptic digestion of protease-resistant inclusion bodies was established. The resulting crude peptide mixture was oxidized by performic acid and the insulin A-chain, which contains no cleavage side for trypsin, was quantified by HPLC in the form of a tetrasulfonate derivative to reduce artefacts due to free -SH groups. Compared with SDS-PAGE, this procedure allows sensitive monitoring of possible degradation at the C-terminus. Furthermore, quantification of expression products on the basis of the present method will provide better correlation between initial and actual yield.
authors.affiliation: Hoechst AG, D-6230-Frankfurt am Main-Höchst-80, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Müllner
authors.forename: V
authors.initials: V
authors.lastname: Hoyle
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D005285:Fermentation; D006801:Humans; D007328:Insulin / Q000032:analysis*; D008722:Methods; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000032:analysis*
chemical_list: D007328:Insulin; D011993:Recombinant Fusion Proteins