Preparation Of Composite Cryogels For Separation Of Plasmid DNA

Supermacroporous monolithic cryogels have large-sized pores of several to several hundreds of microns.Because of their high selectivity and biological compatibility,low mass transfer resistance,rapid adsorption and separation advantages,supermacroporous cryogels are mainly used for the separation and purification of biomacromolecules.Besides these advantages,composite cryogels embedded with particles have good mechanical properties.Stable SiO2 particles with large and uniform size were prepared by optimizing the preparation parameters.Composite polyacrylamide-based matrices embedded with SiO2 particles were obtained.The mechanical strength of the matrices was enhanced by embedding of SiO2 particles.The optimized preparation conditions of the composite matrices were obtained by investigating their properties under embedded with different mass fractions of SiO2.The cation-exchange composite cryogels were prepared by grafting the 2-acrylamido-2-methyl-1-propanesulfonic acid onto the composite matrices,and used to one-step isolation of lysozyme from egg white.The highest purity of lysozyme eluted with 1.5 mol/L NaCl in pH 7.8 sodium phosphate buffer was 81.3%.The anion-exchange cryogels were prepared by grafting diethylaminoethyl-dextran to the epoxide groups of polyacrylamide-based matrices to purify plasmid DNA.The plasmid pUC19 was transferred into Escherichia coli DH5a,cultivated,harvested and lysed.The lysed homogenate was centrifuged and the supernatant was used as the plasmid resource feedstock,which was loaded into the anion-exchange cryogel bed for chromatographic separation.By optimizing the pH of running buffer and the elution conditions,plasmid DNA of 82.8%purity was obtained by elution with 0.5 mol/L NaCl at pH 6.6,with adsorption capacity of 5.4?g/mL cryogel.Compared to the traditional methods for purification of plasmid DNA,animal source enzymes and toxic reagents were not used in the present separation process,ensuring the safety of both the purification operations and the obtained plasmid DNA.