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.
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.
Song
Jae Kwang
JK
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1, Guseong-dong, Yuseong-gu, Daejon 305-701, Korea.
Chung
Bora
B
Oh
Young Hak
YH
Rhee
Joon Shick
JS
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
D008049:Lipase; D010741:Phospholipases A