Study On The Chitosan/cellulose-based Bead Preparation And Application Of Histidine-tagged Protein Purification

With the rapid development of genetic engineering,numerous recombination proteins were obtained using gene recombinant techniques.However,the separation of the desired protein from mixture is a complex and challenging task.It is ongoing to search for new materials and methods of protein separation and purification to meet with the requirement of industrial and commercial processes.In this study,chitosan and cellulose were used as starting materials,using ionic liquid 1-butyl-3-methylimmidazolium chloride([BMIM]Cl)as a solvent,to prepare chitosan/cellulose-based bead(CCB),covering the chitosan's shortage of small powder particles,lack of rigidity,hard to recycle.Subsequently,structures of the resulting beads were well characterized.After binding with metals,the M2+-CCB(M=Ni,Cu,Zn)was further used to the separation and purification of hexahistidine-tagged(his-tagged)proteins expressed in E.coli.The main experimental results are as follows:(1)Uniform spherical shape beads with 3 mm diameter were obtained when the mass ratio of chitosan and cellulose was 4:1.Subsequently,the forming CCB was characterized by fourier transform infrared spectroscopy(FT-IR),the results were attributed to cross-linking between the hydroxyl groups of both cellulose and chitosan through hydrogen bonding.The spherical-shaped and needle-shaped appearance of chitosan and cellulose were observed by transmission electron microscope(TEM).Interestingly,CCB seemed to not only homogeneous but relatively similar with chitosan and cellulose.Furthermore,thermo gravimetric analysis(TGA)showed CCB exhibited good thermal stability under 260 ?.(2)The binding behavior of CCB with metal ions(Ni2+,Cu2+,and Zn2+)was studied.The results indicated that the maximum binding capacity of CCB with Ni2+,Cu2+,and Zn2+ reached 2.38±0.05,3.50±0.08,2.56±0.02 mg/per gram of CCB,respectively,when the concentration of metal ions was 2.4 mg/mL.(3)The affinity adsorption using crude his-tagged EGFP as a target protein were investigated with M2+-CCB(M=Ni,Cu,Zn).The results indicated that the adsorption equilibrium was reached when the amount of adsorbent was 250 mg with constantly vibrating for 30 min.While the optional purification condition was obtained(pH valued 7.2,1.0 mol/L NaCl and the imidazole concentration was 500 mmol/L).It was noted that Zn2+-CCB exhibited the most specific binding ability to the target protein(1.84 mg/g,about 90%purity).(4)The reuse of M2+-CCB(M=Ni,Cu,Zn)adsorbents was investigated under the optimized extraction conditions.The results indicated that the binding capacity of the protein declined quickly with the use times,and the adsorption capacities of Ni2+-CCB,Cu2+-CCB and Zn2+-CCB were 58.7%,53.6%,and 46.9%,respectively,at the forth purification procedure.Additionally,a regeneration method for the adsorbent was further developed by washing with ethylenediaminetetraacetic acid disodium(EDTA)and then re-immobilizing with metal ions.The well adsorption capacities of Ni2+-CCB,Cu2+-CCB,and Zn2+-CCB achieved 97.0%,96.4%,and 97.3%,respectively.(5)The further his-tagged protein separation and purification of bovine serum albumin(BSA)and soybean protein isolate(SPI)was investigated.The results indicated that the purification system developed herein could be applied in the protocol of BSA,however,the high throughout purification of SPI was not feasible.On the other hand,the result showed that separation medium(Zn2+-CCB)had specific adsorption capacity to the histidine.