Pelletier J N, Arndt K M, Plückthun A, Michnick S W
Département de biochimie, Université de Montréal, Québec, Canada.
Nat Biotechnol. 1999 Jul;17(7):683-90. doi: 10.1038/10897.
We describe a rapid and efficient in vivo library-versus-library screening strategy for identifying optimally interacting pairs of heterodimerizing polypeptides. Two leucine zipper libraries, semi-randomized at the positions adjacent to the hydrophobic core, were genetically fused to either one of two designed fragments of the enzyme murine dihydrofolate reductase (mDHFR), and cotransformed into Escherichia coli. Interaction between the library polypeptides reconstituted enzymatic activity of mDHFR, allowing bacterial growth. Analysis of the resulting colonies revealed important biases in the zipper sequences relative to the original libraries, which are consistent with selection for stable, heterodimerizing pairs. Using more weakly associating mDHFR fragments, we increased the stringency of selection. We enriched the best-performing leucine zipper pairs by multiple passaging of the pooled, selected colonies in liquid culture, as the best pairs allowed for better bacterial propagation. This competitive growth allowed small differences among the pairs to be amplified, and different sequence positions were enriched at different rates. We applied these selection processes to a library-versus-library sample of 2.0 x 10(6) combinations and selected a novel leucine zipper pair that may be appropriate for use in further in vivo heterodimerization strategies.
我们描述了一种快速高效的体内文库对文库筛选策略，用于鉴定异源二聚化多肽的最佳相互作用对。在与疏水核心相邻的位置进行半随机化的两个亮氨酸拉链文库，被基因融合到小鼠二氢叶酸还原酶（mDHFR）的两个设计片段之一上，并共转化到大肠杆菌中。文库多肽之间的相互作用重建了mDHFR的酶活性，使细菌得以生长。对所得菌落的分析揭示了拉链序列相对于原始文库的重要偏差，这与选择稳定的异源二聚化对一致。使用结合较弱的mDHFR片段，我们提高了选择的严格性。通过在液体培养中对汇集的、选定的菌落进行多次传代，我们富集了表现最佳的亮氨酸拉链对，因为最佳的配对允许更好的细菌繁殖。这种竞争性生长使得配对之间的微小差异得以放大，并且不同的序列位置以不同的速率富集。我们将这些选择过程应用于一个包含2.0×10⁶种组合的文库对文库样本，并选择了一对可能适用于进一步体内异源二聚化策略的新型亮氨酸拉链。
