Figure 6
From: Directed PCR-free engineering of highly repetitive DNA sequences
Cloning and analysis of the final Poly-Q constructs. (A) The final constructs encode an N-terminal His6-tag (pink) and a SUMO-domain (blue) followed by the 17 N-terminal amino acids of Huntingtin (N17, orange). The different Poly-Q encoding sequences (red) and a FLAG-tag (yellow) are located at the 3' end. Expression is controlled by a T7 promoter (brown). For cloning of the final constructs the Poly-Q encoding repeats of different lengths were excised from of the pMK1-Qn vectors using BsaI and BsmBI. The resulting fragments were inserted into pLANA, which was linearized with BsaI. The restriction sites were arranged in a way that allowed the seamless ligation of the Poly-Q encoding sequences behind N17 into pLANA. Our final constructs still contain a BsaI site behind the Poly-Q encoding region allowing further nucleotide insertions. Used restriction sites are underlined. (B) Three different constructs were successfully produced in E. coli and purified. The proteins consisting of His6-SUMO-N17-Qn-FLAG were applied to SDS-PAGE and visualized by immunoblotting against the FLAG-tag. (C) Purified His6-SUMO-N17-Q47-FLAG fusion proteins were treated with Ulp1 for 1 min and analyzed by SDS-PAGE and α-FLAG immunoblotting. (D) Filter retardation assay using His6-SUMO-N17-Q47-FLAG fusion proteins. Membrane bound Poly-Q fibrils were visualized by α-FLAG immunodetection.