Construction of a General Principle for Increasing Protein ThermostAbility ; Verification of The Proline Theory

这个研究项目的目的是建立一个提高蛋白质热稳定性的一般原理，符合“脯氨酸理论”。从不同温度下生长的3株芽孢杆菌(B. cereus ATCC7064 （I)、B. coagulans ATCC7050 （II）、B. thermoglucosidasius KP1006 (III)）中分别克隆了寡聚- 1,6 -葡萄糖苷酶基因。根据核苷酸序列推断出的同源酶的一级结构进行了比较。结果表明，各酶的脯氨酸残基随热稳定性((I)19-(II)23-(III)32)的增加而增加，且大部分脯氨酸只存在于热稳定性酶（（II）和（III））中，且大部分脯氨酸都包含在环结构中。采用定点诱变的方法，将蜡样芽孢杆菌酶的3个残基（Lys121、Glu175、Glu290）替换为另外两个酶的脯氨酸。Mnt-1=Lys121- P, mnt -2=Lys121- P, Glu175- P, mnt -3=Lys121- P, Glu175- P, Glu290- P)的失活温度分别为44.5,45.9,46.3,47.1,^ C。当每种酶在45 ^ C下处理时，50%失活时间野生为6 min， Mut-1为27 min， Mut-2为120 min，而Mut-3为120 min。这些结果证明，在环上用脯氨酸替代可以有效地提高蛋白质的累积热稳定性，上述原理应该是可行的。有必要确定脯氨酸残基在低聚- 1,6 -葡萄糖苷酶三级结构中的确切位置。为此，在大肠杆菌lac启动子的控制下过量生产蜡样芽孢杆菌酶，并广泛纯化用于结晶和随后的x射线分析。以硫酸铵为沉淀剂，采用悬滴法对酶进行结晶。获得了x射线分析的初步数据，并与大阪大学蛋白质研究所的一个小组合作进行了进一步的分析。少

This research project has been programmed to establish a general principle of increasing protein thermostability, conforming a "proline theory".The genes of oligo-1, 6-glucosidase were cloned individually from three Bacillus strains (B. cereus ATCC7064 (I), B. coagulans ATCC7050 (II), B. thermoglucosidasius KP1006 (III)) growing at a different range of temperature. The primary structures of those homologous enzymes deduced from the nucleotidesequences were determined to be compared. Those revealed that proline residues of each enzyme increased with thermostability ((I)19-(II)23-(III)32) and most of prolines occurred only in thermostable enzymes ((II) and (III)) were included in the loop structures. Three residues (Lys121, Glu175, Glu290) of B. cereus enzyme, substituted by proline in two other enzymes, were replaced with proline by site-directed mutagenesis. The wild and mutant enzymes constructed (Mnt-1=Lys121- P ; Mut-2=Lys121- P, Glu175- P ; Mut-3=Lys121- P, Glu175- P, Glu290- P) sh … More owed that temperatures for 50% inactivation by 10 min holding were 44.5, 45.9, 46.3, 47.1, ^ﾟC, respectively. When each enzyme was treated at 45 ^ﾟC, time for 50% inactivation was 6 min for the wild, 27 min for Mut-1, 120 min for Mut-2, and much longer than 120 min for Mut-3. These results proved that replacements with proline on the loop should be effective to enhance cumulatively thermostability of a protein and the above principle should become practical.It becomes necessary to determine the exact positions of proline residues in the tertiary structure of oligo-1, 6-glucosidase. For the purpose, B. cereus enzyme was overproduced under the control of lac promoter in Escherichia coli and extensively purified for crystallization and subsequen X-ray analysis. The enzyme was crystallized by the hanging drop method using ammonium sulfate as precipitant. The preliminary data of X-ray analysis were obtained and further analysis is going on in collaboration with a group of Institute for Protein Research, Osaka University. Less