Expression, Refolding And Simultaneous Purification Of Recombinant Human Renin Receptor

In recent years, the method of protein folding liquid chromatography (PFLC) has been widely used in protein refolding and purification for its many advantages:denaturant can be rapidly removed and target protein can be efficiently refolded and purified the while, and denaturant can be also recycled by this method. The purpose of this study was to efficiently expressed recombinant human renin receptor (rhRnR) in Escherichia coli (E.coli) at first, and then using 8.0 mol/L urea or 7.0 mol/L guanidine hydrochloride to dissolve them after breaking and washing the rhRnR inclusion bodies which were gained by sonication, centrifugal effect and cleaning solution, finally refolded and simultaneously purified rhRnR by high performance hydrophobic interaction chromatography (HPHIC), high performance ion exchange chromatography (HPIEC) and high performance size exclusion chromatography (HPSEC).The thesis is composed of four sections as follows:1. Literature Review:The methods of protein refolding, the principle and advantages of PFLC were mainly introduced. Physiological and pharmacological functions of RnR, the resreach development of its expression and purification were also introduced in this chapter.2. High expression of rhRnR in E.coli: The impacts of the type of culture media and induce time were investigated for the expression of rhRnR, and the type of denaturants for the refolding effect of rhRnR was also investigated. The results showed that rhRnR had the highest expression when M9 was used as the culture medium and the induced time was 4h, the target protein content in the urea extraction was 7.1 mg/mL; It also showed that using 8.0 mol/L urea as a denaturant would be gained two kinds of rhRnR when it was refolded by HPHIC, which would be its two different molecular conformations or its monomer and dimmer forms, and urea was more favorable for the refolding of it. The purity and mass recovery of the refolded rhRnR were:target protein 1:79.3% and 30.2%; target protein 2: 94.6% and 62.4%, respectively.3. Renaturation and simultaneous purification of rhRnR inclusion bodies by HPIEC:mobile phase conditions (pH, urea concentration), sample volume and surface active agent (also known as detergent) were investigated for the refolding effect of rhRnR. The results showed that only one target protein peak would be gained when rhRnR was refolded by HPIEC, and it could be refolded and simultaneously purified within 30min under optimal condition. Two kinds of rhRnRs were eventually gained when using HPSEC for further purified, which would be its monomer and dimmer forms. After the the flow rate of mobile phase of HPSEC was optimized, the purity and mass recovery of refolded rhRnR were:dimmer, 100% and 12.43%; monomer,100% and 7.82%, respectively, this is helpful to analyze the three-dimensional structure of rhRnR and to research its physiology and pathology. Otherwise, when adding detergent Triton X-100 to the denaturing extract of rhRnR, the finally purity and mass recovery of refolded rhRnR were:dimmer, 92.8% and 62.7%; monomer, 93.5% and 18.3% respectively, this showed that detergent was better to reduce the aggregation of rhRnR and improve its mass recovery.4. Renaturation and simultaneous purification of rhRnR inclusion bodies by HPHIC:Column size, mobile phase conditions (pH, urea concentration) and sample volume were also researched for the refolding effect of rhRnR in this section. It indicated that under optimal conditions, the purity and mass recovery of refolded rhRnR were:target protein 1: 82% and 23.4%; target protein 2:100% and 63%, respectively.