Refolding and purification of recombinant human (Pro)renin receptor from Escherichia coli by ion exchange chromatography.
Purification of the recombinant human renin receptor (rhRnR) is a major aspect of its biological or biophysical analysis, as well as structural research. A simple and efficient method for the refolding and purification of rhRnR expressed in Escherichia coli with weak anion-exchange chromatography (WAX) was presented in this work. The solution containing denatured rhRnR in 8.0 mol/L urea extracted from the inclusion bodies was directly injected into the WAX column. The aggregation was prevented and the soluble form of renatured rhRnR in aqueous solution was obtained after desorption from the column. Effects of the extracting solutions, the pH values and urea concentrations in the mobile phase, as well as the sample size on the refolding and purification of rhRnR were investigated, indicating that the above mentioned factors had remarkable influences on the efficiency of refolding, purification and mass recovery of rhRnR. Under the optimal conditions, rhRnR was successfully refolded and purified simultaneously by WAX in one step within only 30 min. The result was satisfactory with mass recovery of 71.8% and purity of 94.8%, which was further tested by western blotting. The specific binding of the purified rhRnR to recombinant human renin was also determined using surface plasmon resonance (SPR). The association constant of rhRnR to recombinant human renin was calculated to be 3.25 × 10(8) L/mol, which demonstrated that rhRnR was already renatured and simultaneously purified in one step using WAX. All of the above demonstrate that protein folding liquid chromatography (PFLC) should be a powerful tool for the purification and renaturation of rhRnR.
Key Laboratory of Synthetic and Natural Functional Molecule Chemistry of Ministry of Education, Inst. of Modern Separation Science, Key Lab of Modern Separation Science in Shaanxi Province, Northwest University, Xi'an, 710069, China.
Fei
F
Wang
Jinjin
J
Guo
Quan
Q
Bai
Lili
L
Wang
D002852:Chromatography, Ion Exchange; D004926:Escherichia coli; D006801:Humans; D017510:Protein Folding; D058849:Protein Refolding; D011956:Receptors, Cell Surface / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012083:Renin / Q000737:chemistry / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D000093342:Prorenin Receptor
D011956:Receptors, Cell Surface; D011994:Recombinant Proteins; D012083:Renin; D025262:Vacuolar Proton-Translocating ATPases; C458319:ATP6AP2 protein, human; D000093342:Prorenin Receptor
ion exchange chromatography; protein folding liquid chromatography; protein refolding; purification; recombinant renin receptor