title: Expression, purification and characterization of zinc-finger nuclease to knockout the goat beta-lactoglobulin gene.
abstract: Engineered zinc-finger nucleases (ZFNs) have been widely used for precise genome editing. ZFNs can induce DNA double-strand breaks at specific genomic locations and drive the introduction of an insertion or deletion of base pairs at the targeted region, consequently resulting in a loss-of-function mutation. In this study, we investigated the cloning, expression and purification of ZFN fusion proteins targeting the goat beta-lactoglobulin (BLG) gene and detected the cleavage activities of these ZFN proteins in vitro and in cells, respectively. The results showed that the pET-BLG-LFN and pET-BLG-RFN prokaryotic expression plasmids can be constructed correctly and expressed efficiently in Escherichia coli BL21 (DE3) cells to produce the 6× His-tagged ZFN proteins that can be purified by Ni-IDA-Sefinose Column. The predetermined sequence of BLG can be recognized and excised both in vitro and in goat fibroblasts by the purified ZFN fusion proteins, which demonstrated that the purified ZFN fusion proteins can be used as gene modification tools to knock out the BLG gene. Furthermore, these results lay the foundation for eliminating allergen BLG from goat milk and improving the quality of goat milk products.
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Yujie
authors.initials: Y
authors.lastname: Song
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Chenchen
authors.initials: C
authors.lastname: Cui
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Hongmei
authors.initials: H
authors.lastname: Zhu
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Li
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Zhao
authors.affiliation: College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China; Key Laboratory of Animal Biotechnology, Ministry of Agriculture, Northwest A&F University, Yangling, Shaanxi 712100, China. Electronic address: yapingjin1966@yeah.net.
authors.forename: Yaping
authors.initials: Y
authors.lastname: Jin
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D004706:Endodeoxyribonucleases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D055786:Gene Knockout Techniques / Q000379:methods*; D006041:Goats / Q000235:genetics*; D007782:Lactoglobulins / Q000235:genetics*; D019913:Nuclear Localization Signals; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016335:Zinc Fingers
chemical_list: D007782:Lactoglobulins; D019913:Nuclear Localization Signals; D011993:Recombinant Fusion Proteins; D004706:Endodeoxyribonucleases
keywords: Beta lactoglobulin; Direct protein delivery; Fusion protein; Gene targeting; Zinc-finger nucleases