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.

Implications from protein uptake kinetics onto dextran-grafted Sepharose FF coupled with ion exchange and affinity ligands

Our previous studies have reported the presence of "chain delivery" effects of protein adsorption onto ion exchangers with polymer-grafted ion-exchange groups, such as dextran-grafted and poly(ethylenimine)-modified Sepharose gels. However, it is unclear if the "chain delivery" occurs on affinity adsorption with specific interactions. This work is designed to address this issue. A dextran-grafted Sepharose gel was prepared, and then the matrix was modified using diethylaminoethyl, a typical ion-exchange group, or octapeptide(FYCHWQDE), an affinity ligand for human immunoglobulin G(h Ig G) to prepare ion-exchange or affinity adsorbents, respectively.Results of h Ig G adsorption showed that the uptake rate represented by the effective diffusivity of h Ig G onto the dextran-grafted ion exchangers was obviously enhanced by the dextran grafting, indicating the presence of"chain delivery" of the bound proteins on the charged groups on the dextran chains. By contrast, the effective diffusivity of h Ig G changed little as ligand density increased on the dextran-grafted FYCHWQDE adsorbents.Their adsorption capacities decreased and effective diffusivities were not accelerated by the dextran grafting.Thus, this work clarified that grafted dextran could not accelerate h Ig G uptake rate on the affinity resins, or in other words, chain delivery did not occur on the specific interaction-based affinity adsorption.

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.

Study on Separation of Phycoerythrin by Q-Sepharose Fast Flow

[Objective] This study aimed to establish an efficient process for separation of phycoerythrin by using Q Sepharose Fast Flow resin and verity its feasibility for scale-up. [Method] Elution gradient, sample volume and flow rate were optimized to determine the optimal separation condition, under which the scale-up process was verified. [Result] The optimal condition for separation of phycoerythrin by using Q Sepharose FF resin was investigated： 30 ml of laver extract was loaded to the Q Sepharose FF column with a bed volume of 8 ml; subsequently, the column was stepwise eluted with 0-0.10-0.35-1.00 mol/L NaCI solution （pH 6.0） at a constant flow rate of 1 ml/min; the elution peak under 0.35 mol/L NaCI solution was collected, and the recovery rate and purity coefficient （A565/A280） of phycoerythrin were determined as 44.3 and 1.15, respectively. Based on the established process, 75 ml of phycoerythrin extract was loaded to the Q Sepharose FF column with a bed volume of 20 ml for separation, while no significant variation was observed in the separation result. [Conclusion] Phycoerythrin can be well separated from laver extract by using Q Sepharose FF resin and the process is feasible for scale-up.

Separation and purification of Yb_(2)O_(3) by ion exchange chromatography and preparation of ultra-high purity Yb_(2)O_(3)

Ultra-high purity Yb_(2)O_(3) is the critical material of many high-tech materials such as laser glass and fiber,in which impurities seriously affect the laser color quality,intensity and power.In order to reduce the influence of impurities on the properties of laser materials,the purification process of Yb_(2)O_(3) was studied by comparing two kinds of resins(RT-1 and RS-1)using improved ion-exchange chromatography(IEC)method.In this study,through the synergistic improvement of resin structure and eluting system,the environmental pollution caused by ammonia water in the traditional IEC method was reduced,and the requirements of high temperature and pressure were cut.The ion exchange behavior and impurity removal mechanism in the resin column during the loading and eluting process were compared and analyzed.The experimental results show that RS-1 resin is all superior to RT-1resin in elements selectivity,ion exchange capacity and impurities removal rate.After separation and purification by IEC with RS-1 resin,the total removal rate of rare earth impurities was 77.59%and that of non-rare earth impurities was 95.86%when Yb recovery was more than 70%,both higher than that of RT-1 resin(73.26%and 83.18%).This indicates that the improved IEC method is very effective in separating and removing different metal impurities from Yb_(2)O_(3).The pilot test results of IEC method separating and purifying Yb_(2)O_(3) with RS-1 resin show that the purity of Yb_(2)O_(3) can be increased from 99.9929%to 99.9997%by IEC method.It has exhibited huge potential of preparing ultra-high purity Yb_(2)O_(3),especially the deep removal of non-rare earth impurities.

Purification and Stability of an Antithrombus Enzyme from Bacillus subtilis

An antithrombus enzyme (ATE) was precipitated by (NH4)2SO4 or ethanol from supernatant of Bacillus subtilis culture broth then purified using ion exchange chromatography on CM-sepharose fast flow. The effects of ionic strength and pH value on protein adsorption, the gradient elution at different flow rates and step elution were examined respectively. The recovery yield of the optimised process was 74.5% with a purification factor 8.1. The ATE molecular weight was estimated as 30ku by SDS-PAGE. The experimental results showed that the enzyme was stable in the range of pH 7 to pH11, and temperature 25℃ to 37℃.

Retention model of protein for mixed-mode interaction mechanism in ion exchange and hydrophobic interaction chromatography

A unified retention equation of proteins was proved to be valid for a mixed-mode interaction mechanism in ion exchange chromatography (IEC) and hydrophobia interaction chro-matography (HIC). The reason to form a 'U' shape retention curve of proteins hi both HIC and IEC was explained and the concentration range of the strongest elution ability for the mobile phase was determined with this equation. The parameters in this equation could be used to characterize the difference for either HIC or IEC adsorbents and the changes in the molecular conformation of proteins. With the parameters in this equation, the contributions of salt and water in the mobile phase to the protein retention in HIC and IEC were discussed, respectively. In addition, the comparison between the unified equation and Melander' s three-parameter equation for mixed-mode interaction chromatography was also investigated and better results were obtained in former equation.

CHROMATOGRAPHIC REFOLDING OF PROTEINS:MOLECULAR ACTION AND COLUMN CONTROL

Protein expression in E coil often results in the formation of a kind of protein aggregate called inclusion body Conversion of the inactive protein aggregate into biologically active protein is a key step in production of recombinant products Convenlional dilution refolding technique suffers from disadvantages of low recovery and low concentration Various chromatographic refolding techniques have been developed over the last few years These include size-exclusion chromatography, ion exchange chromatography, hydrophobic interaction chromatography and different affinity chromatography. A successful strategy is the use of gradient elution in column control which provides a gentle and gradual change of the solution environment for the macromolecule to rsfold at nano-scale, The gradient refolding at column scale could minimize misfolding and aggregation which are induced by sudden change of the solution in conventional refolding operation.

Influence of microwave on chromium complex composition in tanning liquor

Since microwave irradiation could promote hydrolysis and olation of chromium tanning liquor,but the influence of microwave on chromium complex component in the liquor was still unknown.Chromium sulphate solution(0%basicity)and 33%basicity chromium tanning liquor were subjected microwave(MW)and water bath(WB)heating,and the samples without any warming were regarded as control.Ion exchange chromatography(IEC)and gel filtration chromatography(GFC)were used to measure the charge composition and molecular size of chromium complexes in each sample.FT-IR was used to characterize the structure of chromium complexes in each composition separated by IEC.Moreover,the chromium tanning liquor after warming was used in hide powder tanning trials to illustrate whether microwave would affect its tanning ability.The results show there are more high positive charge and large molecular size complexes in chromium tanning liquor after warming but the phenomena are more significant in MW samples compared with WB due to non-thermal effect of microwave.In addition,microwave has more powerful effect on 33%basicity chromium tanning liquor hydrolysis and olation to generate larger molecular size complexes.In FT-IR results,the combination pattern between chromium and ligands are changed after warming but there is no difference between WB and MW.The chromium exhaustion and thermal stability of hide powder tanned with chromium tanning liquor after microwave irradiation are both higher.It could conclude that both thermal and non-thermal effects of microwave promote the process together,and the nonthermal effect leads to more high positive charge and large molecular size complexes and has stronger influence on high polarity system.In short,this work would provide theoretical basis for applying microwave in tanning agent modification and chrome tanning process further.

"Click chemistry" preparation of WCX packings for protein separation

Click chemistry was applied to immobilize three kinds of alkyne-carboxylic acids onto azide-modified silica gel to prepare three novel stationary phases for weak cation exchange chromatography（WCX）.The developed protocol combines the benefits of operational simplicity,exceptionally mild conditions and high surface loadings.Six kinds of standard proteins were separated completely on the novel packings.Compared with commercial WCX columns,the three kinds of novel WCX packings prepared by click chemistry approach have better resolution and selectivity.Lysozyme was purified successfully from egg white with the novel WCX column by one step.The purity was more than 97%and a high specific activity was achieved to be 81,435 U/mg.The results illustrate the potential of click chemistry for preparation of stationary phase for IEC.

Purification of Murine Monoclonal IgM Antibody

This paper presents the purification of a monoclonal IgM antibody against human tumor associated antigen Lewis\|Y by ion exchange chromatography and gel filtration. Enzyme\|linked immunosorbent assay (ELISA) and SDS\|polyacrylamide gel electrophoresis (PAGE) were used to identify purified IgM antibody. In flow cytometry analysis, the purified IgM antibody recognizes human breast tumor cell line MCF\|7 which expresses Lewis\|Y antigen. This work presents a new way for the purification of murine monoclonal IgM antibody.