재조합 인성장호르몬의 in vitro 풀림과 재접힘 과정의 구조변화 모니터링

Abstract

Using recombinant human growth hormone as a model protein, we carried out unfolding by adding a denaturant such as urea, guanidine HCl, or SDS followed by refolding by dilution and dialysis. The objectives were to monitor the structural changes during in vitro refolding process and, based on the results, to develop a quantitative method of refolding progress assessment. The changes in surface hydrophobicity were measured by fluorescence tagging of 1-anilinonaphthalene-8-sulfonate(1,8-ANS) to the hydrophobic portions, and those in the secondary structure were monitored by using far UV-CD(circular dichroism) spectroscopy. Also, we used RP-HPLC to separate and quantify the folded and unfolded proteins to correlate the result with the structure analysis. Our results indicate the surface hydrophobicity are well correlated with the formations of the secondary structure, primarily α-helices, as well as the disulfide bridges. We expect this monitoring techique can be applied in industrial fields as a means to quantitatively assess the progress of in-vitro refolding of recombinant proteins.