Purification of a viral coat protein by an engineered polyionic sequence.
Virus-like particles composed of the polyoma coat protein VP1 were produced as a central building block of an artificial vector system for gene therapy. For this purpose, recombinant VP1 was expressed in E. coli. Classical purification schemes resulted only in low yields of protein. Therefore, we developed a new affinity purification procedure. We decided to use a polyionic sequence containing eight glutamic acid residues which allows efficient purification using ion-exchange chromatography. This peptide was inserted in a solvent exposed loop on the surface of VP1. After recombinant expression and cell lysis the first purification and concentration step consisted of a fractionated ammonium sulfate precipitation. The resuspended VP1 was loaded on an anion-exchange column. Elution with ca. 600 mM NaCl yielded almost homogeneous protein. Subsequently a size exclusion chromatography was performed to separate the pentameric VP1 from higher oligomeric and aggregated material. In contrast to wildtype VP1 the highly charged mutant form showed no significant tendency to aggregate. To demonstrate the functional state of the VP1 mutant, the in vitro assembly was investigated. At conditions similar to those for wildtype VP1 assembly, the mutant protein could form homogeneous virus-like particles.
Stubenrauch
K
K
Martin-Luther-Universität, Halle-Wittenberg, Institut für Biotechnologie, Halle (Saale), Germany.
Bachmann
A
A
Rudolph
R
R
Lilie
H
H
D001483:Base Sequence; D002213:Capsid / Q000235:genetics / Q000302:isolation & purification* / Q000648:ultrastructure; D002853:Chromatography, Liquid; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D007475:Ion Exchange Resins; D007477:Ions; D008854:Microscopy, Electron; D016296:Mutagenesis; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000648:ultrastructure
D017931:DNA Primers; D007475:Ion Exchange Resins; D007477:Ions; D011994:Recombinant Proteins