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 asthma in 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 asthma vaccine development studies.

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

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

5. Results –Chromatography Results –Chromatography 2 3-1 factorial experiment to explore folding time, effect of ionic buffer and glycerol. Elution Buffer: 100mM Na-Phosphate 10mM TrisHcl 20% v/v Glycerol pH 4.5 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 Pure, refolded Hev b 6 protein

6. Conclusions On-column refolding strategies developed for Hev b 6: from 2 3-1 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.