Refolding of ribonuclease A in vitro and conformational characterization by spectrofluorimetry

Abstract

Ribonuclease A (RNase A) was employed as a model protein to understand the formation of correct three-dimensional structure in the refolding of recombinant proteins in vitro. Important parameters such as the concentration of urea, dilution fold, redox environment (the concentration of GSH and the ratio of GSH to GSSG), pH, temperature, and inhibitor of aggregation were investigated to demonstrate their effects on the activity recovery during refolding. The activity recovery was up to 70% under optimal condition as follows: 100 μg·mL-1 denatured RNase A, refolding buffer containing of 2 mol·L-1 urea, 2 mmol· L-1 GSH and 0.1% PEG6000, the ratio GSH to GSSG 4∶1, pH8.0, shaking speed at 150 r·min-1, 37 ℃. Then, the mechanism of the refolding process of RNase A was characterized using spectrofluorimetry, which results give some supports to presume the conformational changes of RNase A along with the in vitro refolding proceeding.

Keywords

• ribonuclease A
• dilution refolding
• spectrofluorimetry
• molecular structure