Insert engineering and solubility screening improves recovery of virus-like particle subunits displaying hydrophobic epitopes

Abidin, R.S., Lua, L.H.L., Middelberg, A.P.J. and Sainsbury, F. (2015) Insert engineering and solubility screening improves recovery of virus-like particle subunits displaying hydrophobic epitopes. Protein Science, 24 11: 1820-1828. doi:10.1002/pro.2775


Author Abidin, R.S.
Lua, L.H.L.
Middelberg, A.P.J.
Sainsbury, F.
Title Insert engineering and solubility screening improves recovery of virus-like particle subunits displaying hydrophobic epitopes
Journal name Protein Science   Check publisher's open access policy
ISSN 1469-896X
0961-8368
Publication date 2015-11-01
Year available 2015
Sub-type Article (original research)
DOI 10.1002/pro.2775
Open Access Status Not yet assessed
Volume 24
Issue 11
Start page 1820
End page 1828
Total pages 9
Place of publication Hoboken, NJ, United States
Publisher Wiley-Blackwell Publishing
Collection year 2016
Language eng
Abstract The Polyomavirus coat protein, VP1 has been developed as an epitope presentation system able to provoke humoral immunity against a variety of pathogens, such as Influenza and Group A Streptococcus. The ability of the system to carry cytotoxic T cell epitopes on a surface-exposed loop and the impact on protein solubility has not been examined. Four variations of three selected epitopes were cloned into surface-exposed loops of VP1, and expressed in Escherichia coli. VP1 pentamers, also known as capsomeres, were purified via a glutathione-S-transferase tag. Size exclusion chromatography indicated severe aggregation of the recombinant VP1 during enzymatic tag removal resulting from the introduction the hydrophobic epitopes. Inserts were modified to possess double aspartic acid residues at each end of the hydrophobic epitopes and a high-throughput buffer condition screen was implemented with protein aggregation monitored during tag removal by spectrophotometry and dynamic light scattering. These analyses showed that the insertion of charged residues at the extremities of epitopes could improve solubility of capsomeres and revealed multiple windows of opportunity for further condition optimization. A combination of epitope design, pH optimization, and the additive l-arginine permitted the recovery of soluble VP1 pentamers presenting hydrophobic epitopes and their subsequent assembly into virus-like particles.
Keyword Aggregation
Cytotoxic T cell epitope
High-throughput screening
Hydrophobicity
Hydrophobic epitopes
Protein Engineering
Virus-like particles
Q-Index Code C1
Q-Index Status Confirmed Code
Institutional Status UQ

Document type: Journal Article
Sub-type: Article (original research)
Collections: Official 2016 Collection
Australian Institute for Bioengineering and Nanotechnology Publications
 
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Created: Thu, 29 Oct 2015, 12:04:20 EST by Julie Osborne on behalf of Aust Institute for Bioengineering & Nanotechnology