Concanavalin A affinity chromatography for efficient baculovirus purification.
Baculovirus has emerged as a novel gene delivery and vaccine vector, and the demand for purified baculovirus is rising due to the increasing in vivo applications. Since the baculoviral envelope protein gp64 is a glycoprotein, we aimed to develop a concanavalin A (Con A) chromatography process, which harnessed the possible affinity interaction between gp64 and Con A, for simple and effective baculovirus purification. Throughout the purification process the virus stability and recovery were assessed by quantifying the virus transducing titers [TT, defined as transducing units (TU) per milliliter] and viral particles (VP). We found that baculovirus stability was sensitive to buffer conditions and diafiltration with a tangential flow filtration system LabScale using 300 K membranes yielded recoveries of approximately 75% in TT and 82% in VP. The diafiltered baculovirus strongly bound to the Con A column as evidenced by the low virus losses to the flow through and wash fractions. The wash steps eliminated >99% of protein impurities and elution with 0.6 M alpha-D-methylmannoside at room temperature led to the recoveries of approximately 16% in VP and approximately 15.3% in TU. The resultant VP/TU ratio was as low as 41.4, attesting the high quality of the purified virus. Further elution with 1 M alpha-D-methylmannoside recovered another 6% virus TU, yielding a cumulative recovery of approximately 21.3% in TU. These data demonstrated for the first time that Con A chromatography is suitable for baculovirus purification, and may be used for the purification of other viruses with surface glycoproteins.
Chen
Guan-Yu
GY
Department of Chemical Engineering, National Tsing Hua University, Hsinchu 300, Taiwan.
Chen
Chi-Yuan
CY
Chang
Margaret Dah-Tsyr
MD
Matsuura
Yoshiharu
Y
Hu
Yu-Chen
YC
D016367:Baculoviridae / Q000302:isolation & purification* / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D003208:Concanavalin A / Q000737:chemistry* / Q000378:metabolism; D003956:Dialysis; D006863:Hydrogen-Ion Concentration; D008766:Methylmannosides / Q000737:chemistry; D046529:Microscopy, Electron, Transmission; D008970:Molecular Weight; D012965:Sodium Chloride / Q000737:chemistry; D014325:Tromethamine / Q000737:chemistry
D008766:Methylmannosides; D014325:Tromethamine; D003208:Concanavalin A; D012965:Sodium Chloride; C008466:methylmannoside