Preparation Of New Nickel Affinity Chromatographic Medium And Its Application In Isolation Of The Histidine-containing Peptides

Metal chelating affinity chromatography is widely used in the separation and purification of biological macromolecules, especially the purification of histidine tagged proteins. The principle of nickel ion metal chelating affinity chromatography is histidine on the surface of protein coordinate with nickel ion in the medium to achieve the purification and separation.In this paper, the conventional preparation method of nickel ion affinity chromatographic medium was improved. The selection of the carrier, activation method and chelating ligand are based on the IMAC principle. The chloromethylated crosslinking polystyrene microspheres (chlorine ball) was used as the matrix, N,N-Dimethylformamide (DMF) as the solvent and activated by N,N-Diisopropylethylamine (DIEA). The synthesized Lys(Boc)-OEt or Fmoc-Lys-OEt and ethyl chloroacetate was coupled to the matrix. After hydrolysis to release the carboxyl group and chelating with nickel ion, a new nickel ion affinity chromatographic medium was prepared. The affinity medium was applied to purify the His-Lys-Tyr and Phe-His-Thr tri-peptides. The results showed that the histidine-containing peptides were retained to affinity medium and separated with other substance.The nickel ion affinity chromatographic medium was applied to purify the histidine containing No.1 fragment of Exenatide. Establish the best purification system of the No.1 fragment of Exenatide(ASN 1#) under the PBS buffer conditions. The No.2 fragment of Exenatide(ASN2#) which do not containing histidine was used as impurity model. The pH of sample buffer solution, buffer type, imidazole elution and pH buffer elution were screened for isolation of target peptide. After purification of the No.l fragment of Exenatide in various conditions, analysis breakthrough column solution, the balance of the eluent, the imidazole and pH eluting solution by HPLC. The results showed that pH 7.2 was the optimized sample buffer solution. The concentration of imidazole elution was 200 mmol/L. The optimized pH elution buffer solution was pH 3.0 citrate solution. Both citrate solution and imidazole solution can be used to isolate the target peptide and impurity on the nickel ion affinity chromatographic medium. This further proved that the self-made nickel ion affinity chromatographic medium can separate the histidine-containing peptides.