Determination of recombinant Interferon-α2 in E. coli periplasmic extracts by reversed-phase high-performance liquid chromatography.

Reversed-phase high-performance liquid chromatography (RP-HPLC) has been used to analyze Interferon α-2 (IFN-α2) as a pure protein or as a pharmaceutical preparation: a method for analyzing periplasmic IFN-α2 directly in osmotic shock extract has, however, never been reported. This work describes an RP-HPLC methodology for the qualitative and quantitative analysis of human IFN-α2a and IFN-α2b directly in bacterial periplasmic extracts or in purified preparations. The analytical method has been set up and validated for accuracy, precision, linearity, sensitivity and specificity. A recovery test indicated an average bias of ∼1%, intra-day and inter-day quantitative determinations presented relative standard deviations always≤5%, while the working sensitivity was of ∼0.3µg of IFN-α2 (RSD=5%). The method proved to be suitable for detecting and quantifying also glycosylated and oxidized forms and N-methionylated IFN-α2 molecules, it was, however, not able to distinguish between IFN-α2a and IFN-α2b. This rapid methodology allows the application of RP-HPLC as a powerful tool to monitor the production yield and quality of IFN-α2 in osmotic shock fluids, right after, or even during the fermentation process.

Expression, purification, and characterization of authentic mouse prolactin obtained in Escherichia coli periplasmic space.

Prolactin (PRL) is a pleiotropic hormone produced by lactotroph cells of the anterior pituitary gland and is mainly related to lactation control and reproduction. Recombinant mouse prolactin (r-mPRL), never obtained in its authentic form, can be very useful for research and tests in animal models, in which human prolactin (hPRL) is usually employed in a heterologous mode. Synthesis of r-mPRL was carried out here via secretion in Escherichia coli periplasmic space using a plasmid containing mPRL cDNA joined to the DsbA signal peptide sequence under the control of a constitutive major leftward promoter of the bacteriophage λ (λPL). Fermentation in a pilot bioreactor was carried out at 30°C, with 6 H of induction at 37°C, reaching an optical density of 23 A600 units, a specific yield of 0.06-0.1 µg mPRL/(mL A600), and a concentration of up to 2.2 µg/mL. Even with such a low yield and a poor mass fraction, r-mPRL was purified via a three-step laboratory process based on hydrophobic chromatography, reversed-phase high-performance liquid chromatography, and high-performance size-exclusion chromatography (HPSEC). The purified hormone was then characterized using SDS-PAGE, Western blotting, and HPSEC and showed, by Nb2 rat lymphoma cell proliferation assay, a bioactivity of 39.5 IU/mg, determined against the International Standard of recombinant hPRL [World Health Organization (WHO)-97/714].