Scaling-up fermentation of Escherichia coli for production of recombinant P64k protein from Neisseria meningitidis

Background: P64k is a Neisseria meningitidis high molecular weight protein present in meningococcal vaccine preparations. The lpdA gene, which encodes for this protein, was cloned in Escherichia coli and the P64k recombinant protein was expressed in E. coli K12 GC366 cells under the control of a tryptophan promoter. P64k was expressed as an intracellular soluble protein about 28% of the total cellular protein. Several scale-up criteria of fermentation processes were studied to obtain the recombinant P64k protein at the pilot production scale. Results: The best operational conditions at a larger scale production of P64k recombinant protein were studied and compared using the four following criteria: Constant Reynold's number (Re constant), Constant impeller tip speed (n di constant), Constant power consumption per unit liquid volume (P/V constant) and Constant volumetric oxygen transfer coefficients (KLa/k constant). The highest production of the recombinant protein was achieved based on the constant KLa/k scale-up fermentation criterion, calculating the aeration rate (Q) and the impeller agitation speed (n) by iterative process, keeping constant the KLa/k value from bench scale. The P64k protein total production at the 50 l culture scale was 546 mg l -1 in comparison with the 284 mg l -1 obtained at 1.5 l bench scale. Conclusions: The methodology described herein, for the KLa/k scale-up fermentation criterion, allowed us to obtain the P64k protein at 50 l scale. A fermentation process for the production of P64k protein from N. meningitidis was established, a protein to be used in future vaccine formulations in humans.

Preliminary Research on the Milk Allergy Induced by High Molecular Weight Protein / 天津医药

Objective To investigate the allergization of milk high molecular weight proteins. Methods Thirty cas?es of patients with serum allergic to milk were selected. Their skin prick tests were positive. Results of serum specific IgE (sIgE) test were positive and≥1+. One case of healthy control with negative sIgE test and without history of allertgy, was in?cluded in this study. The serum samples were collected and frozen at-20℃. Sephadex G200 gel chromatography was used to obtain milk high molecular weight proteins. Western blot and ELISA methods were used to detect milk high molecular weight proteins and the activity of serum sIgE. Results Results of SDS-PAGE showed that high molecular weights proteins displayed by Sephadex G200 gel chromatography, mainly including three bands, the molecular weight of 67 ku, 80 ku and 160 ku. Western blot analysis showed that three kinds of high molecular weights proteins can react with milk allergy serum, and the most obvious appeared near the molecular weight 67 ku band. ELISA analysis showed that the positive response rate of high molecular weight proteins with milk allergic patient’s serum was slightly higher than that ofβ-lactoglobulin (46.7%and 43.3%, respectively). Conclusion The milk high molecular weight protein components can induce specific IgE antibod?ies, which have important sensitization in the process of milk allergy.