Expression, purification and characterization of zinc-finger nuclease to knockout the goat beta-lactoglobulin gene

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 6x 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. (C) 2015 Elsevier Inc. All rights reserved.