A novel protein refolding method integrating ion exchange chromatography with artificial molecular chaperone

Artificial molecular chaperone （AMC） and ion exchange chromatography （IEC） were integrated, thus a new refolding method, artificial molecular chaperone-ion exchange chromatography （AMC-IEC） was developed. Compared with AMC and IEC, the activity recovery of lysozyme obtained by AMC-IEC was much higher in the investigated range of initial protein concentrations, and the results show that AMC-IEC is very efficient for protein refolding at high concentrations. When the initial concentration of lysozyme is 180 mg/mL, its activity recovery obtained by AMC-IEC is still as high as 76.6%, while the activity recoveries obtained by AMC and IEC are 45.6% and 42.4%, respectively.

STUDY OF DERIVATIZATION OF PROTEIN WITH A EUROPIUM CHELATE AND APPLICATION TO HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

A europium chelate of 5-chlorosulfoyl-2- thenoyltrifluoroacetone(CTTA)was investigated for precolumn derivatization of protein for high performance liquid chromatography.This new label was highly fluorescent and suitable for time-resolved fluorometric detection.The detective limit of protein is less than 0.3ug in sample with a volume of 100ul.Theoretically,the new label can also be used in gel electrophoresis and protein blotting.

Protein adsorption onto diethylaminoethyl dextran modified anion exchanger:Effect of ionic strength and column behavior

Our previous studies on bovine serum albumin （BSA） adsorption to diethylaminoethyl dextran （DEAE dextran, DexD, grafting-ligand） and DEAE （D, surface-ligand） modified Sepharose FF resins found that all the grafted resins （FF-DexD and FF-D-DexD） exhibited extremely fast uptake rate （effective diffusivity, De, De/Do 〉 1.4）, which was six times greater than the ungrafted resins （De/Do 〈 0.3）. In this work, the influence of ionic strength （IS） on 6 typical DEAE dextran-grafted resins was investigated. Bath adsorption equilibria and kinetics, breakthrough, and linear gradient elution experiments were conducted. Commercial DEAE Sepharose FF was used for comparison. It is found that protein adsorption capacities on DEAE dextran-FF resins and the commercial resin decreased with increasing IS, but DEAE dextran-FF resins exhibited much higher capacity sensitivity to salt concentration. Besides, steeper decrease of adsorption capacities could be obtained at higher graftingligand or surface-ligand density. It is worth noting that the facilitating role of surface-ligand to the ＂chain delivery＂ effect was weakened after adding salt, leading to the less improvement in uptake rate by increasing surface-ligand density at higher IS. Although the uptake rates of the DEAE dextran-FF resins increased first and then decreased with increasing fS, they kept the extremely high level of De values （De/Do 〉 1.1 ） at the their working/binding IS range. Moreover, the DEAE dextran-FF resin displayed much higher adsorption capacities and De values than commercial ungrafted resin in their working condition. Furthermore, the column results of DEAE dextran-FF resins presented higher dynamic binding capacities than and similar elution ISs with DEAE Sepharose FF to achieve similar （or even higher） recoveries suggest the excellent chromatographic column performance of the DEAE dextran-FF resins. Finally, both high recovery and purity of BSA and γ-globulin could be easily achieved using the typical DEAE dextran-FF column, FF-D60-DexD160, to separate their binary mixtures, by step gradient elution. The research has provided new insights into the practical application of the series of DEAE-dextran grafted resins in protein chromatography and proved their superiority.

Preparation of Supermacroporous Composite Cryogel Embedded with SiO_2 Nanoparticles

Supermacroporous composite cryogels embedded with SiO2 nanoparticles were prepared by radical cryogenic copolymerization of the reactive monomer mixture of acrylamide（AAm） and N,N-methylene-bis-acrylamide（MBAAm） containing SiO2 nanoparticles（mass ratios of nanoparticles to the monomer AAm from 0.01 to 0.08） under the freezing-temperature variation condition in glass columns.The properties of these composite cryogels were measured.The height equivalent to theoretical plate（HETP） of the cryogel beds at different liquid flow rates was determined by residence time distribution（RTD） using tracer pulse-response method.The composite cryogel matrix embedded with the mass fraction of SiO2 nanoparticles of 0.02 presented the best properties and was employed in the following graft polymerization.Chromatographic process of lysozyme in the composite cryogel grafted with 2-acrylamido-2-methyl-1-propanesulfonic acid（AMPSA） was carried out to evaluate the protein breakthrough and elution characteristics.The chromatography can be carried out at relatively high superficial velocity,i.e.,15 cm·min-1,indicating the satisfactory mechanical strength due to the embedded nanoparticles.