New Guanidine-based Protein Folding Chromatographic Stationary Phase And The Refolding Of Rhg-csf Purification Research

Protein refolding is a research hot point in downstream of biotechnology.In this paper, two kinds of chromatographic stationary phase coupled with arginine and creatine were synthesized.The argirtine-type stationary phase was intensively investigated and employed for the refolding of the recombinant human granulocyte colony-stimulating factor(rhG-CSF) which containing two pairs of disulfide bond.This article includes four sections:1 Literature review.It contines the significance and methods,mainly for protein folding liquid chromatography(PFLC) and its related technology.The contribution of arginine and stationary phase to protein refolding and the recent researchs of refolding and purification of rhG-CSF were also briefly reviewered.It contains 58 references.2 Two kinds of silica-based stationary phase coupled with arginine or creatine were synthesized.With optimizational condition and tested with standard proteins,the arginine-type stationary phase was found to be better.3 With the separation of five kinds of acidic protein under different pH and different flow rate,the mass recovery of the five proteins were found totally greater than 95%.When BSA was selected as the model protein,its maximum adsorbed amout of the arginine-type stationary phase was measured＞46.1mg/g packings.These results indicate that the stationary phase has the typical character of anion exchange chromatography and has good column capacity and small non-specific adsorption.4 Using arginine-type chromatographic stationary phase to do refolding and purification of rhG-CSF expressed in E.coli.The experiments were carried out on different gradient,pH, urea concentration and flow rate.With an optimizational condition,the finally obtained rhG-CSF had purity of 95.24%and mass recovery of 68%by only one step in 30 min.The source of mass loses was found to be both of unretard of rhG-CSF as sample injection(～10%) and the rest as some precipitated on the stationary phase which did not dissolved as it was eluted with 4 mL,0.5mol/L NaCl,8mol/L urea and 20mmol/L DTT.