title: Construction of DNA-shuffled and incrementally truncated libraries by a mutagenic and unidirectional reassembly method: changing from a substrate specificity of phospholipase to that of lipase.
abstract: A method of mutagenic and unidirectional reassembly (MURA) that can generate libraries of DNA-shuffled and randomly truncated proteins was developed. The method involved fragmenting the template gene(s) randomly by DNase I and reassembling the small fragments with a unidirectional primer by PCR. The MURA products were treated with T4 DNA polymerase and subsequently with a restriction enzyme whose site was located on the region of the MURA primer. The N-terminal-truncated and DNA-shuffled library of a Serratia sp. phospholipase A(1) prepared by this method had an essentially random variation of truncated size and also showed point mutations associated with DNA shuffling. After high-throughput screening on triglyceride-emulsified plates, several mutants exhibiting absolute lipase activity (NPL variants) were obtained. The sequence analysis and the lipase activity assay on the NPL variants revealed that N-terminal truncations at a region beginning with amino acids 61 to 71, together with amino acid substitutions, resulted in the change of substrate specificity from a phospholipase to a lipase. We therefore suggest that the MURA method, which combines incremental truncation with DNA shuffling, can contribute to expanding the searchable sequence space in directed evolution experiments.
authors.affiliation: Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejon 305-701, Korea.
authors.forename: Jae Kwang
authors.initials: JK
authors.lastname: Song
authors.forename: Bora
authors.initials: B
authors.lastname: Chung
authors.forename: Young Hak
authors.initials: YH
authors.lastname: Oh
authors.forename: Joon Shick
authors.initials: JS
authors.lastname: Rhee
mesh_terms: D032621:DNA Shuffling / Q000379:methods*; D015723:Gene Library; D008049:Lipase / Q000235:genetics* / Q000378:metabolism; D016296:Mutagenesis; D010741:Phospholipases A / Q000235:genetics* / Q000378:metabolism; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods*; D012705:Serratia / Q000201:enzymology / Q000235:genetics*; D013379:Substrate Specificity
chemical_list: D008049:Lipase; D010741:Phospholipases A