Refolding And Simultaneous Purification Of The Recombinant Human Flt3 Ligand By Protein Folding Liquid Chromatography

Protein folding liquid chromatography (PFLC) is increasingly being applied to the refolding and simultaneous purification of inclusion body proteins in recent years. The characteristics of PFLC are that it can not only separate the target protein from other constituents, but also make denatured protein folding in the column. In this dissertation,①the shaking flask culture conditions of recombinant human Flt3 ligand (rhFL) expressed in E.coli were studied, and high-level expressed rhFL inclusion bodies were obtained.②The cells were disrupted by ultrasonication, purified inclusion bodies were colleceted after centrifugation and repeated washing and were dissolved in 8 mol/L urea, obtaining the denatured protein extracts.③Retention feature and renaturation rules of rhFL in the process of refolding and simultaneous purification were investigated by using high performance hydrophobic interaction chromatography (HPHIC) method, in which an analytical HPHIC column, a semi-preparative HPHIC cake and a novel two-dimensional liquid chromatography by a single column were separately employed, this provided a foundation for large scale preparation of rhFL protein and its clinical research applications.The dissertation consists of four sections:1. Literature review:The recent advance and applications of PFLC methods with different mechanisms in the protein refolding and simultaneous purification were reviewed, and the FL structure, biological functions, its importance in clinical applications were also reviewe.2. Optimization of conditions for expressing rhFL in the shaking flask:-Influence of culture media in the shaking flask, culture conditions (temperature, inducing time, pH, inoculation amount) on the expression of rhFL were investigated. The experimental results showed that when the culture temperature was 35℃, initial pH of SOB culture medium was 6.6, glycerol was selected as a carbon source, inducing time was 4 h, inoculation amount was 4%, the expression amount of rhFL protein could be up to 47.4% of the total bacterial protein. 3. Refolding and simultaneous purification of rhFL from inclusion bodies expressed in E.coli by HPHIC:Chromatographic columns with different dimensions (analytical chromatographic column, semi-preparative chromatographic cake) were separately used, effects of chromatographic mobile phase (small molecular additives, flow rate) and loading volume on the mass recovery of rhFL during the refolding and purification were investigated, the optimum conditions and retention rules of the renaturation and purification of rhFL on HPHIC column were obtained, fluorescence intensity variations of rhFL inclusion bodies before and after the refolding in the chromatographic column were preliminarily investigated by fluorescence spectrometry, and rhFL was prepared in the large scale by using HPHIC cake. The results showed that the mass recovery of rhFL could reach to 45.9% and 67.0% respectively after refolded and purified by using the analytical chromatographic column and the semi-preparative chromatographic cake, both being packed with stationary phase with phenyl ligands, the purity were 93.5% and 92.1% respectively. The activity results of cell proliferation on G-CSF dependent cell line NFS-60 showed that the average specific bioactivity of rhFL was 7.42×108 IU/mg when synergized with G-CSF.4. Refolding and simultaneous purification of rhFL inclusion bodies by a novel two-dimensional liquid chromatography:2DLC-1C comumn, which was a novel single column with both HIC and IEC retention models, has been employed for fast separation of active proteins. In this work, a 2DLC-1C column was used in HIC mode to investigate effect of salt concentration in the mobile phase on the renaturation and simultaneous purification of rhFL, and the results were compared with a chromatographic column with phenyl ligands. On the chromatographic column with phenyl ligands, when the (NH4)2SO4 concentration in the mobile phase was 3.0 mol/L, the mass recovery of rhFL was 48.7%, while the mass recovery was 48.5% on the 2DLC-1C column when the (NH4)2SO4 concentration in the mobile phase was 2.0 mol/L, which shows that a relatively high mass recovery can be obtained on the 2DLC-1C column at lower salt concentration. This makes it possible to reduce cost in preparation of rhFL.