Combinatorial protein engineering by incremental truncation
Combinatorial protein engineering by incremental truncation
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DOI：
10.1073/pnas.96.7.3562
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发表时间：
1999-03-30
影响因子：
11.1
通讯作者：
Benkovic, SJ
中科院分区：
文献类型：
--
作者：
Ostermeier, M;Nixon, AE;Benkovic, SJ
我们开发了一种组合方法，使用重叠的N端和c端基因片段的增量截断文库，检查酶的给定区域内所有可能的平分点，这些平分点将允许单体酶转化为其功能异源二聚体。这种酶对分的通用方法将在通过交换结构域和化学合成修饰肽片段的新催化功能工程以及酶进化和蛋白质折叠的研究中具有广泛的应用。我们在大肠杆菌甘氨酸酰胺核糖核苷酸甲酰转移酶（PurN）上测试了这种方法，并通过遗传选择鉴定出能够进行甘氨酸酰胺核糖核苷酸转化的PurN异源二聚体。选择了两种进行物理表征，并发现在变性稳定性，活性以及底物和辅因子的结合方面与野生型PurN单体相当。对18个随机选择的活性PurN异源二聚体的序列分析显示，断点主要聚集在酶表面附近的环中，断裂可能导致高度保守残基的缺失，最令人惊讶的是，活性位点可以被分割。
We have developed a combinatorial approach, using incremental truncation libraries of overlapping N- and C-terminal gene fragments, that examines all possible bisection points within a given region of an enzyme that will allow the conversion of a monomeric enzyme into its functional heterodimer. This general method for enzyme bisection will have broad applications in the engineering of new catalytic functions through domain swapping and chemical synthesis of modified peptide fragments and in the study of enzyme evolution and protein folding. We have tested this methodology on Escherichia coli glycinamide ribonucleotide formyltransferase (PurN) and, by genetic selection, identified PurN heterodimers capable of glycinamide ribonucleotide transformylation. Two were chosen for physical characterization and were found to be comparable to the wild-type PurN monomer in terms of stability to denaturation, activity, and binding of substrate and cofactor. Sequence analysis of 18 randomly chosen, active PurN heterodimers revealed that the breakpoints primarily clustered in loops near the surface of the enzyme, that the breaks could result in the deletion of highly conserved residues and, most surprisingly, that the active site could be bisected.