Refolding Experiment Using "Loose Folding Method" That Induces Correct 3D Structure from Denatured Protein

(1)利用变性还原的鸡蛋清溶菌酶进行了复性实验。活动的范围是ca。在Lyzm浓度为10 μ M时，通过简单稀释法从6 M氯化胍（GdmCl）溶液中获得20%。未回收的酶的大部分导致聚集。在0.5-1 M GdmCl存在下，蛋白质分子预期处于松散重折叠状态，恢复的活性急剧增加高达80%。用尿素和LiCl的混合溶剂代替GdmCl，可使活性恢复100%。(2)如乙二醇-LiCl和甘油-LiCl的组合溶剂也带来了变性和还原的Lyzm的完美恢复，可能是由于实现了蛋白质分子的松散重折叠状态。(3)对于灰色链霉菌胰蛋白酶（SGT）的复性，三乙醇胺或二乙醇胺（1-2 M）是最佳的介质，而对Lyzm或枯草杆菌蛋白酶BPN ′的复性有效的尿素-LiCl或乙酸钾对SGT的复性则没有帮助。发现对于蛋白酶的复性，除了选择复性之外，诸如防止自溶的附加装置保证了回收率的增加，培养基诱导变性蛋白质分子的松散重折叠状态。

(1) Refolding experiment was carried out by use of denatured and reduced hen egg-white lysozyme (Lyzm). Recovered activity was ca. 20% at the Lyzm concentration of 10 muM by simple dilution method from 6 M guanidinium chloride (GdmCl) solution. The major part of the unrecovered enzyme resulted in aggregation. In the presence of 0.5-1 M GdmCl where the protein molecule is expected to be in loosely refolded state, the recovered activity increased drastically up to 80%. Using combined solvent of urea and LiCl instead of GdmCl, the recovered activity of 100% could be attained.(2) Combined solvents such as ethylene glycol-LiCl and glycerol-LiCl also brought about the perfect recovery of the denatured and reduced Lyzm probably due to realization of the loosely refolded state of protein molecule.(3) For the refolding of Streptomyces griseus trypsin (SGT) the solution of triethanolamine or diethanolamine at 1-2 M revealed to be excellent medium whereas urea-LiCl or potassium acetate effective to the refolding of Lyzm or subtilisin BPN', respectively was not useful to the refolding of SGT.Furthermore, it was found that for the refolding of the protease such an additive device as prevention of autolysis guaranteed the increase of the recovery yield beside the selection of refolding media to induce the loosely refolded state of denatured protein molecules.