title: Production of Well-Characterized Virus-like Particles in an Escherichia coli-Based Expression Platform for Preclinical Vaccine Assessments.
abstract: In this chapter we demonstrate a method to produce virus-like particles (VLPs) from Escherichia coli. Standard bacterial protocols are used for the cloning, transformation, and expression of the protein subunits. A two-step protein purification method is highlighted: one step based on separating soluble proteins with ion-exchange affinity chromatography and a second polishing step using size-exclusion columns to isolate VLP species. The ensuing VLPs can be characterized with a variety of biophysical techniques including ultraviolet (UV)-visible spectroscopy for protein quantification, dynamic light scattering for size distribution determination, and transmission electron microscopy to ascertain size and morphology.
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, 2095 Constant Ave, Lawrence, KS, 66047, USA. wahome@ku.edu.
authors.forename: Newton
authors.initials: N
authors.lastname: Wahome
authors.affiliation: Protein Production Group, University of Kansas, Lawrence, KS, 66047, USA.
authors.forename: Anne
authors.initials: A
authors.lastname: Cooper
authors.affiliation: Microscopy and Analytical Imaging Lab, University of Kansas, Lawrence, KS, 66047, USA.
authors.forename: Prem
authors.initials: P
authors.lastname: Thapa
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, 2095 Constant Ave, Lawrence, KS, 66047, USA.
authors.forename: Shyamal
authors.initials: S
authors.lastname: Choudhari
authors.affiliation: Protein Production Group, University of Kansas, Lawrence, KS, 66047, USA.
authors.forename: Fei P
authors.initials: FP
authors.lastname: Gao
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, 2095 Constant Ave, Lawrence, KS, 66047, USA.
authors.forename: David B
authors.initials: DB
authors.lastname: Volkin
authors.affiliation: Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, 2095 Constant Ave, Lawrence, KS, 66047, USA.
authors.forename: C Russell
authors.initials: CR
authors.lastname: Middaugh
mesh_terms: D036022:Capsid Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004353:Drug Evaluation, Preclinical; D000067493:Dynamic Light Scattering; D004926:Escherichia coli / Q000235:genetics*; D005818:Genetic Engineering / Q000379:methods*; D046529:Microscopy, Electron, Transmission; D013056:Spectrophotometry, Ultraviolet; D014170:Transformation, Genetic; D058425:Vaccines, Virus-Like Particle / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D036022:Capsid Proteins; D058425:Vaccines, Virus-Like Particle
keywords: Dynamic light scattering; Expression; Purification; Transmission electron microscopy; Ultraviolet (UV)–visible absorbance spectroscopy; Virus-like particles