Ion-exchange membrane chromatography method for rapid and efficient purification of recombinant baculovirus and baculovirus gp64 protein.
Significant progress in the application of baculoviral vectors for gene delivery into mammalian cells calls for the development of powerful methods for virus purification and concentration. We report here a novel and efficient method based on membrane chromatography to prepare baculoviral stocks. On a strong cation-exchange membrane unit, baculovirus in insect cell culture supernatants was captured at a flow rate of 3 ml/min and efficiently eluted at the same flow rate with a physiological buffer containing 150 mM NaCl as a desorption reagent. The procedure allowed for a final recovery of 78% of infective viral particles from the original supernatant and 30-fold enrichment. The high purity of the viral preparation was demonstrated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. Baculovirus gp64 proteins could be purified by the same method, indicating the importance of the protein in mediating the binding of baculovirus to the cation-exchange membrane. The method developed should be suitable for preparing baculoviral stocks, and probably other gp64-pseudotyped viral vectors, for gene therapy applications.
Wu
Chunxiao
C
Institute of Bioengineering and Nanotechnology, Singapore.
Soh
Ker Yin
KY
Wang
Shu
S
D000818:Animals; D016367:Baculoviridae / Q000254:growth & development / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005822:Genetic Vectors / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000302:isolation & purification*; D012680:Sensitivity and Specificity; D018411:Spodoptera / Q000821:virology; D013997:Time Factors; D014763:Viral Matrix Proteins / Q000302:isolation & purification*; D015678:Viral Structural Proteins / Q000302:isolation & purification*
D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; D015678:Viral Structural Proteins; C065417:major structural protein, cytomegalovirus 64kDa