Construction of stabilized proteins by combinatorial consensus mutagenesis
Construction of stabilized proteins by combinatorial consensus mutagenesis
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DOI：
10.1093/protein/gzh091
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发表时间：
2004-11-01
影响因子：
2.4
通讯作者：
Schellenberger, V
中科院分区：
文献类型：
--
作者：
Amin, N;Liu, AD;Schellenberger, V
我们通过组合引入共有突变构建了阴沟肠杆菌β -内酰胺酶（BLA）的稳定变体。通过比对38种BLA同源物的序列，我们确定了阴沟肠杆菌基因与内酰胺酶共有序列存在差异的29个位点，并使用涵盖所有29个位点的诱变寡核苷酸混合物构建了组合文库。对从这些文库中随机选取的90个分离株进行筛选，鉴定出15个热稳定性显著提高的变体。这些分离株的稳定性表明，所有测试的突变对蛋白质稳定性都有累加贡献。对序列和稳定性数据的统计分析确定了11个具有稳定作用的突变和8个使蛋白质不稳定的突变。将这11个稳定突变重新组合构建的第二代文库，使我们鉴定出稳定性进一步提高的BLA变体。最稳定的变体其热变性中点（Tm）比起始分子高9.1℃，并且包含8个共有突变。将3个稳定的BLA变体与几种蛋白酶一起孵育，结果表明所有测试的分离株对蛋白水解的抗性都显著提高。我们的数据表明，组合共有诱变（CCM）能够快速产生热稳定性和抗蛋白水解稳定性提高的蛋白质变体。
We constructed stabilized variants of beta-lactamase (BLA) from Enterobacter cloacae by combinatorial recruitment of consensus mutations. By aligning the sequences of 38 BLA homologs, we identified 29 positions where the E.cloacae gene differs from the consensus sequence of lactamases and constructed combinatorial libraries using mixtures of mutagenic oligonucleotides encompassing all 29 positions. Screening of 90 random isolates from these libraries identified 15 variants with significantly increased thermostability. The stability of these isolates suggest that all tested mutations make additive contributions to protein stability. A statistical analysis of sequence and stability data identified 11 mutations that made stabilizing contributions and eight mutations that destabilized the protein. A second-generation library recombining these 11 stabilizing mutations led to the identification of BLA variants that showed further stabilization. The most stable variant had a mid-point of thermal denaturation (T-m) that was 9.1degreesC higher than the starting molecule and contained eight consensus mutations. Incubation of three stabilized BLA variants with several proteases showed that all tested isolates have significantly increased resistance to proteolysis. Our data demonstrate that combinatorial consensus mutagenesis (CCM) allows the rapid generation of protein variants with improved thermal and proteolytic stability.