Multiple approaches for the establishment of the basis of Protein Engineering.

我们已经完成了如下所述的各种研究，以建立“蛋白质工程”的基础。1、替换氨基酸的方法：(1)证实T4酶内切酶的Glu23残基对糖基酶活性有重要影响。(2)发现H^+ atp酶的Thrl56残基对激活活性起重要作用。(3)通过检测NADHcytochrome b_5的结构-功能关系，了解遗传性高铁血红蛋白血症的病因。(4)溶菌酶中Trp108对诱导活性的作用明显。2、构建目标蛋白表达体系的方法：(1)牛组织因子的结合位点存在于凝血因子VII中Gal和EGF结构域之间的区域。(2)阐明了枯草芽孢杆菌淀粉样芽孢杆菌中性蛋白酶的活性位点及其催化机制。(3)分析了嗜热脂肪芽孢杆菌l -赖氨酸和ATP与赖氨酸tRNA合成酶的结合方式。(1)赖氨酸肽酶HIs210残基在高选择性激活活性中起重要作用。(2)分析了哈布蛇磷脂酶A_2的结构与功能的关系。(3)通过制备醛缩酶的各种嵌合蛋白，分析了醛缩酶的催化机理。(4)通过对三磷酸异构酶和核糖核酸酶的模块结构分析，探讨了它们的结构、功能和遗传特性。4、化学修饰方法：(1)通过引入新型聚乙二醇和直链淀粉衍生物，使RNase T1和胰蛋白酶稳定。(1)对长双歧杆菌l -乳酸脱氢酶的变构现象进行了分析。(2)利用核磁共振和距离几何算法确定了neocarzinostatatin的三级结构。(3)通过与胰蛋白酶配合物的x射线晶体学分析，考察了Bowman-Birk蛋白酶抑制剂的功能。少

We have accomplished various Investigations described below for the establishment of the basis of 'Protein Engineering'.1, The approach by replacing amino acid : (1) Glu23 residue of T4 endonuclease was proved to be Important in glycosylase activity. (2) Thrl56 residue of H^+ ATPase was found to be important to evoke activity. (3) The structure-function relationship of NADHcytochrome b_5 was examined to understand the cause of hereditary methemoglobinemia. (4) The role of Trp108 in lysozyme on evoking activity became apparent.2, The approach by constructing expression system of aimed protein : (1) The binding site of bovine tissue factor was present in the region between Gal and EGF domain in blood coagulation factor VII. (2) The active sites and the catalytic mechanism of neutral protease from Bacillus subtilis var. amylosacchariticus were elucidated. (3) The binding mode of L-lysine and ATP with lysyl tRNA synthase from Bacillus stearothermophilus were analyzed.3, The approach by com … More paring primarx and tertiary structure of proteins : (1) HIs210 residue of lysylendopeptidase was proved to be important in high-selectivity on evoking activity. (2) The relationship between structure and function in phospholipase A_2 from Habu snake was analyzed. (3) The catalytic mechanism of human aldolase was analyzed by preparing its various chimeric proteins. (4) The structure, function and heredity of both triosephosphate isomerase and ribonuclease were examined discussed by the analysis of their module structures.4, The approach by means of chemical modification : (1) RNase T1 and trypsin became stable by introducing novel polyethyleneglycol and amylose derivative.5, The approach by use of NMR and X-ray crystallography : (1) The allosteric phenomena of L-lactic acid dehydrogenase from Bifidobacterium longum was analyzed. (2) The tertiary structure of neocarzinostatin was determined by use of NMR and distance geometry algorism. (3) The function of Bowman-Birk protease inhibitor was examined from the X-ray crystallographic analysis of its complex with trypsin. Less