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Towards an Asthma Vaccine: Scalable Process for the Purification of a Latex Allergen Protein

Towards an Asthma Vaccine: Scalable Process for the Purification of a Latex Allergen Protein. Presented by : Anushi E. Kulasiri. The Problem. 80% of occupational asthma in the healthcare industry is caused by latex allergies.

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Towards an Asthma Vaccine: Scalable Process for the Purification of a Latex Allergen Protein

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  1. Towards an Asthma Vaccine: Scalable Process for the Purification of a Latex Allergen Protein Presented by : Anushi E. Kulasiri

  2. The Problem • 80% of occupational asthmain the healthcare industry is caused by latex allergies. • 70 – 86% of healthcare workers affected by latex allergies is caused by Hev b 6. • Researchers need large amounts of pure, biologically active Hev b 6 protein for asthmavaccine development studies.

  3. Project Goals Enhanced production of pure, biologically active Hev b 6protein by improving: • Protein release from E.coli cells • Protein solubalization • On-column refolding

  4. Schematic Process Flow Diagram for Method Fermentation Sonication Centrifugation Chromatography For removal of cell debris and some contaminants Cells lysis via ultrasound to release inclusion bodies from E.Coli. Hev b 6 protein over expression in E. Coli Target protein isolated and refolded and contaminants removed. Further analysis and experimentation for latex allergenicity vaccine project.

  5. Lysis Buffer: 8M Urea, 100mM Na- Phosphate, 10mM TrisHcl, pH 8 Wash Buffer: 8M Urea, 100mM Na-Posphate, 10mM TrisHcl, pH 6.3 Refolding Buffer: 100mM Na-Phosphate, 10mM TrisHcl, pH 6.3 Elution Buffer: 100mM Na-Phosphate 10mM TrisHcl 20% v/v Glycerol pH 4.5 Results –Chromatography23-1 factorial experiment to explore folding time, effect of ionic buffer and glycerol. Pure, refolded Hev b 6 protein

  6. Conclusions • On-column refolding strategies developed for Hev b 6: from 23-1 factorial experiment Time showed to have largest effect on protein elution. • Successful purification of soluble Hev b 6. • High pressure homogenization shown to be a feasible method to increase yield.

  7. Acknowledgements • My supervisor, Gareth M. Forde • My lab partner, Ronny • Ellen, Prathab, Susan and Jenny • Alec Drew at CRC for Asthma • Work for this project was funded by the Monash NMSR Grant and the Summer Research Experience Scholarship Program.

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