Analysis of the higher-order structure formation from the amino terminus of protein

构建了一种新的直接表达载体ATG-TAG载体，并应用于鸡溶菌酶模块组合1+2+3+4+5（以下简称M1 -5，依此类推）、M1 -4、M1 -3、M2-5和M2-4在大肠杆菌中的表达。未进行Mi-2和Ml作为融合蛋白的表达。在非诱导期，基本培养基对表达有抑制作用，而丰富培养基对表达无抑制作用。迄今为止所使用的IPTG浓度以及培养温度的变化并不导致表达成可溶性级分。表达产物的一级结构与预期一致。当二硫键桥打开时，这些模块组合显示出CD光谱，其表明与真正的天然溶菌酶相比，α-螺旋减少，β-折叠含量增加。这种“残留”结构表现出对盐酸胍诱导的变性的非合作转变。甘油、山梨醇、甲醇和三氟乙醇增加了 ...更多信息 简化模块组合的二级结构。除M1 -5和M2-4外，复性（再氧化）模块组合包含十种以上不同高级结构的分子种类，如通过RPHPLC监测的。当在多元醇或醇的存在下再氧化时，M2-5显示出高阶结构的增加。然而，没有观察到三级结构的合作形成。另一方面，缺少一个二硫键而保留全长多肽的溶菌酶衍生物在甘油存在下被有效地复性，并显示出与真实溶菌酶相当的裂解活性和二级结构。另一种衍生物，其中包含残基21至129，并可以形成三个天然的二硫键，没有显示出一个“正确的”折叠，即使在甘油的存在下。总之，氨基端（残基1 - 20）和羧基端（残基108-129）是鸡溶菌酶正确形成高级结构所必需的。少

A new direct expression vector, ATG-TAG vector, was developed, and applied to the expression in Escherichia coli of hen lysozyme module combinations 1+2+3+4+5(hereafter abbreviated as Ml-5, and so on), Ml-4, Ml-3, M2-5 and M2-4. The expression of Mi-2 and Ml as fused proteins was not carried out. The repression of expression during uninduced period was observed when minimal media were used, but was not when rich media were used. Variation in IPTG concentration as well as in the culture temperature so far used did not lead to the expression into a soluble fraction. The primary structures of the expressed materials were as expected. These module combinations, when disulfide-bridges were opened, showed the CD spectra which indicated a decrease in alpha-helix and an increase in beta-sheet contents compared to authentic native lysozyme. This 'residual' structure showed a non-cooperative transition on GuHCl-induced denaturation. Glycerol, sorbitol, methanol and trifluoroethanol increased the … More secondary structure of the reduced module combinations. Renatured(reoxidized)module combinations contained, ' except for Ml-5 and M2-4, more than ten molecular species of different higher-order structure as monitored by RPHPLC. M2-5 showed an increase in higher-order structure when reoxidized in the presence of polyols or alcohols. Cooperative formation of tertiary structure, however, was not observed. on the other hand, the lysozyme derivatives which lacked one disulfide bond while retaining fulllength polypeptide were renatured efficiently in the presence of glycerol, and showed lytic activities and secondary structures comparable to those of authentic lysozyme. Another derivative which contained residues 21 to 129, and which could form three native disulfide bonds, did not show a 'correct' folding even in the presence of glycerol. Altogether, both amino-terminal(residues 1 to 20)and carboxyl-terminal region(108-129)are necessary for the correct higherorder structure formation of hen lysozyme. Less