Purification of capsid-like particles of infectious bursal disease virus (IBDV) VP2 expressed in E. coli with a metal-ion affinity membrane system.
Protein VP2, matured from the polyprotein encoded by the genome of infectious bursal disease virus (IBDV) and the primary host-protective immunogen of this virus, together with its two N-terminal truncated mutants were cloned and expressed in Escherichia coli. To obtain pure recombinant proteins for the development of an efficient subunit vaccine against IBDV infection, these three proteins were fused with six additional histidine residues at their C-termini as a His-purification-tag. Following purification employing immobilized metal-ion (Ni2+) affinity chromatography (IMAC), a purification fold of approximately 104 was achieved. Electron microscopic observation also demonstrated that all three E. coli-derived proteins form the morphology of icosahedral particles of approximately 25 nm in diameter. To reduce the cost of resin used for IMAC, self-prepared immobilized metal-ion affinity membranes (IMAM), i.e., commercial, regenerated cellulose membrane modified with iminodiacetic acid and immobilized with nickel ions, was applied to purify particles formed by these three proteins. A 104-fold of purification efficiency was also achieved by this membrane, showing that under the same conditions the recovery and purification efficiency of IMAM are comparable with those of IMAC. The pure VP2-formed particles thus obtained, coupled with their uniform dimensions, not only facilitate a better understanding of the structural biology of these immunogenic particles but also help the development of improved vaccines against this avian virus.
Chen
Chien-Shiuh
CS
Department of Chemical Engineering, National Chung Hsing University, Taichung, Taiwan 40227, ROC.
Suen
Shing-Yi
SY
Lai
Su-Yuan
SY
Chang
Gary Ro-Lin
GR
Lu
Tsung-Chi
TC
Lee
Meng-Shiou
MS
Wang
Min-Ying
MY
D002482:Cellulose; D002846:Chromatography, Affinity / Q000379:methods*; D004926:Escherichia coli / Q000378:metabolism*; D007477:Ions; D009532:Nickel; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000096:biosynthesis; D015678:Viral Structural Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*
D007477:Ions; D011994:Recombinant Proteins; C082513:VP2 protein, infectious bursal disease virus; D015678:Viral Structural Proteins; D009532:Nickel; D002482:Cellulose