Transcript Towards an Asthma Vaccine: Scalable Process for the

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.
• 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.
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
Schematic Process Flow
Diagram for Method
Fermentation
Hev b 6 protein
over expression
in E. Coli
Sonication
Cells lysis via
ultrasound to
release
inclusion bodies
from E.Coli.
Centrifugation
Chromatography
For removal of
Target protein
cell debris and
isolated and
some
refolded and
contaminants
contaminants
removed.
Further analysis
and
experimentation
for latex
allergenicity
vaccine project.
Results –Chromatography
23-1 factorial experiment to explore folding time,
effect of ionic buffer and glycerol.
Lysis Buffer:
Wash Buffer:
8M Urea,
100mM Na- Phosphate,
10mM TrisHcl,
pH 8
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
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.
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.