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

(1)采用变性还原蛋清溶菌酶（Lyzm）进行复折叠试验。从6 M氯化胍（GdmCl）溶液中采用简单稀释法，在10 muM的Lyzm浓度下，回收率约为20%。未回收酶的主要部分导致聚集。在0.5-1 M GdmCl存在时，蛋白质分子预计处于松散折叠状态，恢复活性急剧增加，最高可达80%。用尿素- LiCl复合溶剂代替GdmCl，可达到100%的回收率。(2)乙二醇- licl和甘油- licl等复合溶剂也能使变性和还原的Lyzm得到完美的恢复，这可能是由于实现了蛋白质分子的松散折叠状态。(3)对于灰色链霉菌胰蛋白酶（SGT）的重折叠，1 ~ 2 M的三乙醇胺或二乙醇胺溶液是较好的培养基，而尿素- licl或醋酸钾分别对Lyzm或枯草菌素BPN'的重折叠无效。研究发现，对于蛋白酶的重折叠，除了选择重折叠介质诱导变性蛋白分子的松散重折叠状态外，防止自解等附加装置保证了回收率的提高。

(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.