A method for directly determining intact recombinant insulin fusion proteins in crude fermentation extracts
✍ Scribed by Stefan Müllner; Vivienne Hoyle
- Publisher
- Elsevier Science
- Year
- 1992
- Tongue
- English
- Weight
- 587 KB
- Volume
- 202
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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.