课件：酶的分子修饰backgrou.ppt

1990年Goodson将干扰素的Thr3替换为Cys3，并通过PEG修饰，水溶性增加，半衰期延长(工程二硫键 ) 噬菌体T4溶菌酶Ile3突变为Cys3，使与Cys97形成二硫键，催化活性不变，但热稳定性显著提高(工程二硫键) xylanase(工程二硫键) used to treat wood pulp in paper production needs to function at high temp Glu49位于色氨酸合成酶的疏水核心部分。如Ala取代，稳定性增加(增加蛋白质内部疏水性) Three-Dimensional Structure of T4 Lysozyme How many residues can be systematically changed and retain the same fold? or what fraction of the sequence is necessary for folding? To assess how many residues are necessary or essential for maintaining the same fold, single and multiple alanines were inserted into three helices in T4 lysozyme. 39-50; 115-123; and 126-134. This type of approach is opposite to designing a protein from first principles. What are the observations and conclusions? Location of new disulfide bonds in T4 lysozyme Introduction of disulfide bonds must be formed in the context of a structure. Only a limited number of locations in a protein can accept two cysteine residues in an orientation that can accept the S-S bond without generating conformational strain (software now exists which can help in this choice). In principle, the larger the loop the greater the reduction in the entropy of the unfolded state. Lys取代葡萄糖脱氢酶的Ser231，突变体在55℃ 的半衰期比天然酶高8倍，活力相当 (酶表面亲水化) 枯草杆菌蛋白酶的Cys取代Met222催化速度增加2倍；用Ala、Ser 或Leu取代Met222，酶可以抵抗高达1mol/L H2O2的氧化失活 (抗氧化失活) 三糖磷酸异构酶的Asn144用Thr取代，Asn78用Ile取代，突变酶在100℃ 的稳定性比天然酶高2倍；枯草杆菌α-淀粉酶Asn190用Ala取代，在80℃ 的半衰期增加6倍(Asn脱酰胺失活) PCR定点突变(PCR mutagenesis) Introduces sequence changes into (cloned) DNA fragments; Overlap extension method requires 2 mutagenic primers and 2 others; Amplify a 5′ fragment and a 3′ fragment that overlap and each have the mutation; Use these products in another reaction to produce the full-length mutated DNA. PCR mutagenesis- overlap extension SP6 primer forward mutagenic primer reverse mutagenic primer T7 primer X x x x Two separate 1st PCRs remove primers, denature and re-anneal 2nd PCR “If a biochemist wishes to improve on a be