Aqueous two-phase systems as an alternative process route for the fractionation of small inclusion bodies.
Aqueous two-phase protocols have been established which successfully generate highly purified preparations of small inclusion bodies (IBs) from whole cell homogenates. Particle size analysis of disruptates confirmed that intense disruption (concomitant with maximal product release) was compromised by the corelease of contaminating solutes and the micronisation of cell debris yielding a similar particle size range to the IBs (100-200 nm). PEG 300-phosphate systems enabled partial recovery of IBs in the top phase of ATPS. In contrast, PEG 8000-phosphate systems partitioned IBs more efficiently as a discrete sediment within the lower phase, whilst the majority of micronised debris remained in the interphase. The alpha-glucosidase IB yield and purity in ATPS was bettered only by analytical sucrose density gradient centrifugation, which is not readily scaleable for application in process operations. The successful recovery of such small IBs from complex homogenates highlights a generic role that ATPS techniques might play in the recovery and purification of new bioparticulate products (viral and plasmid gene therapy vectors, particulate protein vaccines etc.).
Walker
S G
SG
Biochemical Recovery Group, Centre for Bioprocess Engineering, School of Chemical Engineering, University of Birmingham, Edgbaston, UK.
Lyddiatt
A
A
D002458:Cell Fractionation / Q000379:methods*; D002498:Centrifugation; D002499:Centrifugation, Density Gradient; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry; D005285:Fermentation; D005374:Filtration; D002479:Inclusion Bodies