A method for directly determining intact recombinant insulin fusion proteins in crude fermentation extracts.
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.
Müllner
S
S
Hoechst AG, D-6230-Frankfurt am Main-Höchst-80, Germany.
Hoyle
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V
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*
D007328:Insulin; D011993:Recombinant Fusion Proteins