O'Loughlin Taryn L, Matsumura Ichiro
Department of Biochemistry, Center for Fundamental and Applied Molecular Evolution, Emory University School of Medicine, Rollins Research Center, Room 4119, 1510 Clifton Road, Atlanta, GA 30322, USA.
Comb Chem High Throughput Screen. 2006 May;9(4):313-20. doi: 10.2174/138620706776843219.
Our long-term goal is to direct the evolution of novel protease variants. To this end we have engineered a new type of protease-activated reporter enzyme. Many protease-activated enzymes evolved in nature, but the introduction of novel regulatory mechanisms into normally unregulated enzymes poses a difficult design challenge. Random Elongation Mutagenesis [1] was used to fuse the p6 peptide, which is recognized and cleaved by HIV protease, and twelve random sequence amino acids to the C-termini of beta-glucuronidase (GUS) and alkaline phosphatase (AP). The resulting GUS-p6-(NNN)12 and AP-p6-(NNN)12 libraries were expressed in E. coli and screened for clones that were inactivated by the C-terminal extension (tail). The inactivated clones were co-expressed with HIV protease, and those that were re-activated were isolated. The AP and GUS activities of the most responsive clones were each >3.5-fold higher when co-expressed with HIV protease, and this activation is correlated with in vivo proteolysis. It should be possible to generalize this strategy to different reporter enzymes, different target proteases, and perhaps to other types of protein-modifying enzymes.
我们的长期目标是引导新型蛋白酶变体的进化。为此，我们设计了一种新型的蛋白酶激活报告酶。自然界中进化出了许多蛋白酶激活酶，但将新型调控机制引入通常无调控的酶中带来了艰巨的设计挑战。随机延伸诱变[1]被用于将可被HIV蛋白酶识别并切割的p6肽以及十二个随机序列氨基酸融合到β-葡萄糖醛酸酶（GUS）和碱性磷酸酶（AP）的C末端。所得的GUS-p6-(NNN)12和AP-p6-(NNN)12文库在大肠杆菌中表达，并筛选因C末端延伸（尾巴）而失活的克隆。将失活的克隆与HIV蛋白酶共表达，并分离出重新激活的克隆。与HIV蛋白酶共表达时，反应最灵敏的克隆的AP和GUS活性均高出>3.5倍，且这种激活与体内蛋白水解相关。将该策略推广到不同的报告酶、不同的靶蛋白酶，甚至可能推广到其他类型的蛋白质修饰酶应该是可行的。
