Expression, Refolding And Purification Of RHIV-1 Vif 158

Protein refolding liquid chromatography (PFLC) in the process of refolding recombinant protein expressed in E.coli as inclusion bodies can removing denaturant fastly, purifing and refolding target protein, recycling denaturant, it is widely applied to proteins refolding and purification. In this dissertation,①the culture conditions of recombinant human immunodeficiency virus type one virion infectivity factor 158 (rHIV-1 Vif 158) expressed in E.coli were studied, and highiy expressed rHIV-1 Vif 158 inclusion bodies were obtained under the optimal culture conditions;②the cells were disrupted by ultrasonication, the inclusion bodies were colleceted after washing with cleaning buffer solution, the solubilized conditions were studied, and the extract solution of inclusion bodies can be obtained;③rHIV-1 Vif 158 in the proeess of refolding and simultaneous purification were investigated by using PFLC, which is very important to research the structure and function of rHIV-1 Vif 158.The dissertation consists of four sections:1. Literature review:The progress of rHIV-1 Vif structure and function were introduced. The significance of rHIV-1 Vif 158 and protein refolding was introduced briefly. The principles and applications of PFLC methods in the process of protein refolding and purification were reviewed.2. The optimization of the conditions for expression rHIV-1 Vif 158 and solubilization rHIV-1 Vif 158 inclusion bodies:The influence of culture media in shake flask, culture temperature, inducing time on the expression of rHIV-1 Vif 158 and the solubilized conditions for solubilizing the rHIV-1 Vif 158 inclusion bodies were investigated. The experimental results showed that when the culture media was M9 medium, culture temperature was 35℃, inducing time was 4 hour, the expression amount of rHIV-1 Vif 158 protein can be obtained 33.1% of the total bacterial protein. The low concentrations of urea combined with detergent dissovle the rHIV-1 Vif 158 inclusion bodies can obtained better results. The 5 mol/L urea containing 1% Sodium Lauroyl Sareosine (SLS) as the best condition for the solubilizing rHIV-1 Vif 158 inclusion bodies.3. Refolding and purification of rHIV-1 Vif 158 inclusion bodies by HPHIC:The effects of the extract solution, urea concentions and pH value in the mobile phase, gradient conditions, loading volume on the renaturation and stimultaneous pruification of rHIV-1 Vif 158 with HPHIC were investigated. The results showed that at the optimum condition the mass recovery and purity can be obained to be 48.6% and 91.3%, respectively.4. Refolding and purification of rHIV-1 Vif 158 inclusion bodies by SEC: The effects of different urea concentration in SEC and the flow rate in urea gradient SEC of rHIV-1 Vif 158 on the refolding and pruification was investigated. The results showed that the mass recovery of rHIV-1 Vif 158 could reach to 42.7% and 55.3% respectivety after refolded and purified by using SEC and urea gradient SEC, the purity were 86.4% and 89.3%, respectively.