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

Content Provider	World Health Organization (WHO)-Global Index Medicus
Author	Lua, L. H. L. Sainsbury, F. Abidin, R. S. Middelberg, A. P. J.
Description	Country affiliation: Australia Author Affiliation: Abidin RS ( The University of Queensland, Australian Institute for Bioengineering and Nanotechnology Centre for Biomolecular Engineering, St Lucia, Queensland, 4072, Australia.); Lua LH ( Protein Expression Facility, The University of Queensland, St Lucia, Queensland, 4072, Australia.); Middelberg AP ( The University of Queensland, Australian Institute for Bioengineering and Nanotechnology Centre for Biomolecular Engineering, St Lucia, Queensland, 4072, Australia.); Sainsbury F ( The University of Queensland, Australian Institute for Bioengineering and Nanotechnology Centre for Biomolecular Engineering, St Lucia, Queensland, 4072, Australia.)
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.
ISSN	09618368
e-ISSN	1469896X
Journal	Protein Science
Issue Number	11
Volume Number	24
Language	English
Publisher	Wiley-Blackwell (on behalf of The Protein Society)
Publisher Date	2015-11-01
Publisher Place	United States
Access Restriction	Open
Subject Keyword	Epitopes Chemistry Genetics Protein Engineering Virion High-throughput Screening Assays Hydrophobic And Hydrophilic Interactions Solubility Research Support, Non-u.s. Gov't Discipline Biochemistry
Content Type	Text
Resource Type	Article
Subject	Medicine Molecular Biology Biochemistry

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