The addition of packing material for high performance hydrophobic interaction chromatograghy (HPHIC) into the denaturant solution to prevent, or depress protein aggregation in the denatuaration process is presented. The renaturation of α-chymotrypsin (α-Chy) denatured with guanidine hydrochloride (GuHCl) solution indicated that renaturation efficiency can be enhanced from 36.1% to 59.0% by this new method. The structure of the ligand linking of HPHIC packings is also important for the protein renaturation.
Ye Hua SHEN Quan BAI Yang Jun ZHANG Yin Mao WEI Hai Bo WANG Xing Du GENG
A procedure for the preparation of continuous rod consisting of poly(glycidyl methacrylate\|co\|ethylene dimethacrylate) for immobilized metal affinity chromatography (IMAC) is presented. When chelating Cu(Ⅱ), Zn(Ⅱ) or Ni(Ⅱ) on it, the rod displayed a property of IMAC and the selectivity for protein separation was different from that obtained from the naked rod. An abnormal increase in retention times of proteins was found under the condition of either very high pH or very low pH on the metal chelated rods.
Tile extract of E.coli containing recombinant human inteferon- (rhIFN-) with 7.0 mol/L guanidine hydrochloride ((Gu’IICl) was directly injected into a column of reverse phase liquid chromatography (RPLC) to separate and purify rhIFN- with acetic acid-water as mobile phase. Gu.HCI and most impure protcins call be separated by this way. Compared with the usual dilution method, the bioactivity recovery of tile purified rhIMN- was found to be over 500%. In addition. compared to common organic solvents employed in RPLC, acetic acid has higher freezing point. and therefore, it is easy to concentrate tile aim-protein by freeze-drying when acetic acid-water is used as Inobile phase in RPLC.
