Siau Jia Wei, Chee Sharon, Makhija Harshyaa, Wai Cho Mar Myint, Chandra Shree Harsha Vijaya, Peter Sabrina, Dröge Peter, Ghadessy Farid J
p53 Laboratory, 8A Biomedical Grove, #06-06, Immunos, Singapore 138648, Singapore.
School of Biological Sciences, Nanyang Technological University, 60 Nanyang Drive, Singapore 637551, Singapore.
Protein Eng Des Sel. 2015 Jul;28(7):211-20. doi: 10.1093/protein/gzv015. Epub 2015 Mar 18.
Advances in genome engineering are attendant on the development of novel enzyme variants with programed substrate specificities and improved activity. We have devised a novel selection method, wherein the activity of a recombinase deletes the gene encoding an inhibitor of an enzyme conferring a selectable phenotype. By using β-lactamase and the β-lactamase inhibitor protein, the selection couples recombinase activity to Escherichia coli survival in the presence of ampicillin. Using this method, we generated λ integrase variants displaying improved in vitro recombination of a non-cognate substrate present in the human genome. One generalist integrase variant displaying enhanced catalytic activity was further used in a facile, single-step transformation method to introduce transgenes up to 8.5 kb into the unique endogenous attB site of common laboratory E.coli strains.
基因组工程的进展伴随着具有编程底物特异性和改进活性的新型酶变体的开发。我们设计了一种新颖的筛选方法，其中重组酶的活性删除了编码赋予可选择表型的酶抑制剂的基因。通过使用β-内酰胺酶和β-内酰胺酶抑制剂蛋白，该筛选将重组酶活性与氨苄青霉素存在下大肠杆菌的存活联系起来。使用这种方法，我们生成了λ整合酶变体，其在体外对人类基因组中存在的非同源底物的重组表现出改进。一种显示出增强催化活性的通用整合酶变体进一步用于一种简便的单步转化方法，将长达8.5 kb的转基因引入常见实验室大肠杆菌菌株的独特内源性attB位点。
