Affinity capture of a biotinylated retrovirus on macroporous monolithic adsorbents: towards a rapid single-step purification process.
A streptavidin derivitised macroporous monolith was developed to enable single-step capture of chemically biotinylated Moloney Murine Leukaemia Virus (MoMuLV) from crude, unclarified cell culture supernatant. Monoliths were prepared by aqueous cryopolymerisation of acrylamide with N,N''-methylene-bis (acrylamide) and glycidyl methacrylate (Arvidsson et al. [2003] J Chrom A 986:275-290). Streptavidin was immobilised to the epoxy functionalised monoliths. Particulate-containing cell culture supernatant was passed through the monolith without preclarification of the feedstock and adsorption capacities of 2 x 10(5) cfu/ml of adsorbent were demonstrated (cf. Fractogel streptavidin, at 3.9 x 10(5) cfu/ml of adsorbent). The specific titre of the recovered fraction was increased by 425-fold; however, recoveries of less than 8% were achieved. Adsorption of nonbiotinylated MoMuLV on the streptavidin-coated monolith was not observed.
Williams
Sharon L
SL
University of Cambridge, Department of Chemical Engineering, New Museums Site, Pembroke Street, Cambridge, Cambridgeshire, CB2 3RA, United Kingdom.
Eccleston
Mark E
ME
Slater
Nigel K H
NK
D000178:Acrylamides / Q000737:chemistry; D000327:Adsorption; D000818:Animals; D019921:Biotinylation; D002846:Chromatography, Affinity / Q000379:methods*; D003470:Culture Media; D005240:Feasibility Studies; D006367:HeLa Cells; D006801:Humans; D008689:Methacrylates / Q000737:chemistry; D051379:Mice; D008979:Moloney murine leukemia virus / Q000302:isolation & purification / Q000378:metabolism; D041681:NIH 3T3 Cells; D049329:Nanostructures; D011108:Polymers; D012190:Retroviridae / Q000302:isolation & purification* / Q000378:metabolism*; D019809:Streptavidin / Q000378:metabolism; D014867:Water / Q000737:chemistry
D000178:Acrylamides; D003470:Culture Media; D008689:Methacrylates; D011108:Polymers; D014867:Water; D019809:Streptavidin