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https://hdl.handle.net/2440/106652
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Type: | Journal article |
Title: | Insert engineering and solubility screening improves recovery of virus-like particle subunits displaying hydrophobic epitopes |
Author: | Abidin, R. Lua, L. Middelberg, A. Sainsbury, F. |
Citation: | Protein Science, 2015; 24(11):1820-1828 |
Publisher: | Wiley-Blackwell |
Issue Date: | 2015 |
ISSN: | 0961-8368 1469-896X |
Statement of Responsibility: | R. S. Abidin, L. H. L. Lua, A. P. J. Middelberg, and F. Sainsbury |
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. |
Keywords: | Aggregation; cytotoxic T cell epitope; high-throughput screening; hydrophobicity; hydrophobic epitopes; protein aggregation; protein engineering; virus-like particles |
Description: | Published online 24 September 2015 proteinscience.org |
Rights: | © 2015 The Protein Society. Published by Wiley-Blackwell. |
DOI: | 10.1002/pro.2775 |
Grant ID: | http://purl.org/au-research/grants/arc/DE140101553 |
Published version: | http://dx.doi.org/10.1002/pro.2775 |
Appears in Collections: | Aurora harvest 3 Chemical Engineering publications |
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