HRV-16 capsid proteins (VP1, VP2, and VP0) have been successfully ... Human rhinovirus (HRV) infections are one of the most significant risk factors for asthma.
An Efficient Approach for Recombinant Expression and Purification of Rhinovirus-16 Capsid Proteins in E. coli Sayeh Agah1, James Hindley2, Martin Chapman1, and Sabina Wünschmann1
# 358
INDOOR Biotechnologies Inc., Charlottesville, VA. USA1 INDOOR Biotechnologies LTD. Cardiff, UK2
Rationale Human rhinovirus (HRV) infections are one of the most significant risk factors for asthma exacerbations leading to acute care visits. Surface exposed capsid proteins (VP1, VP2, and VP3) are important for binding of HRV to human epithelial cells. These proteins are insoluble and individually unstable, making purification extremely difficult. Here we describe an efficient method for expression and purification of HRV-16 capsid proteins VP1, VP2, and VP0 (VP2 + VP4) in E. coli. The purified capsid proteins provide useful tools to study the immune mechanisms involved in rhinovirus-induced asthma exacerbations, epitope mapping, and diagnostic purposes.
•
VP1, VP2, and VP3 are naturally intertwined, and very unstable individually, making purification of the individual proteins extremely difficult. VP1, VP2, VP3 are not soluble, and remain in inclusion bodies.
•
VP2
VP4
•
Molecular weight
HRV-16 VP1
33 kDa (Runs at ~37kDa On SDS-PAGE)
HRV-16 VP2
29 kDa
HRV-16 VP3
26 kDa
HRV-16 VP0 (VP2 + VP4)
39 kDa
VP1
HRV-16 genome poly protein
VP3 VP4
•
Expression in E. coli Rosetta 2 cells.
•
Purification from the insoluble fraction.
•
Purification of each protein individually.
• VP3
VP3
Asymmetric unit
Biological assembly
VP2
• HRVs attach to cellular receptors such as ICAM-1, which promotes vial uncoating and release of viral RNA into cells. • ICAM-1 interacts mainly with VP1 and VP2. • HRV-16 capsid proteins are good targets for a potential HRV vaccine. • There are no reliable, high quality sources of the protein available on the market. Kolatkar et. Al., (1999) The EMBO Journal Vol.18 No.22 pp.6249–6259
LDLR
Purification of VP0 (VP2 + VP4) as a single polypeptide due to expected increased stability, and interest in research projects.
37 25
37 25
VP2
VP0 (VP2 + VP4) VP2
VP3 VP4 Anti-VP2
Anti-His tag
Anti-VP2
VP0 •
HRV-16 capsid proteins (VP1, VP2, and VP0) have been successfully expressed, and purified to high purity and have been validated by western blot.
endosome
VP0 (VP2 + VP4)
B
A
Results ICAM1
Lot generated
VP1
HRV-16 capsid assembly
Biology of HRV Capsid Proteins
Validated (by western blot)
Validation of HRV-16 VP2 and HRV-16 VP0 by western blot.
VP2
VP2
Purified
Expression and Purification Strategy
VP1
VP2
VP4
VP3
Protein
VP1
VP0
Viral capsid is composed of 60 copies of each of the four proteins, VP1, VP2, VP3, and VP4. Epitopes for antibody binding are on VP1, VP2 and VP3.
VP1
VP2
List of purified HRV-16 capsid proteins.
Non-Structural Proteins
Capsid Proteins
HRV16 Capsid Proteins •
Results (continued)
Why so Difficult?
A)
50 37 25
VP1
B)
37
25
VP2
C)
50 37 25
VP0
D)
VP3
37
25
Host epithelial cell SDS-PAGE coomassie and silver stained analysis of A) VP1,and coomassie stained B) VP2, C) VP0, and D) VP3.
Western blot of VP2 probed by A) anti-VP2 mAb and B)
•
Western blot of VP0 probed by anti-VP2 mAb
anti-His tag antibody
CONCLUSIONS • The best purification strategy consists of purification of the capsid proteins in 8M urea over NiNTA resin followed by refolding by a 2 step dialysis, and a final gel filtration chromatography. • Due to instability of VP3 alone, we aim to produce a VP0-VP3 fusion protein. This will cover the entire HRV-16 capsid. • The purified HRV-16 capsid proteins are applicable to research on HRV infections, HRV vaccines, and immune mechanisms involved in rhinovirus-induced asthma exacerbations.
