Expression, Purification, Crystallization, and X-ray Structural Analysis of CRISPR-Associated Protein Cas6 from Methanocaldococcus jannaschii.

Ming-Chang Lee; Shih-Ting Tseng; Juan-Cheng Yang; Tung-Ju Hsieh; Shang-Chuen Wu; Shu-Min Kuan; Ming-Jen Chen; Ming-Chiu Chang; Chun-Chiu Wang; Hsiu-Lin Chen; Guor-Cheng Fang; Winn-Jung Huang; Tzu-Ping Ko; Yeh Chen

The CRISPR-associated protein 6, Cas6 protein, is an endoribonuclease that cleaves precursor CRISPR RNAs within the repeat sequence to release specific invader-targeting RNAs. Cas6 protein can recognize different sequences by their specific scaffold. To investigate its binding mode, we purified and crystallized a His-tagged Cas6 protein from Methanocaldococcus jannaschii (MjCas6) using the sitting-drop vapor-diffusion method. The crystals diffracted to a resolution of 1.85 Å and belonged to monoclinic space group C2, with unit-cell parameters a = 200.84 Å, b = 85.26 Å, c = 100.06 Å, β = 118.47°. The crystals of MjCas6 contain four molecules in the asymmetric unit. The protein fold is similar to the other Cas6 homologues, such as Pyrococcus furiosus Cas6, suggesting functional similarity. Moreover, in the C2 crystal the MjCas6 monomers formed a tandem array, which we hypothesize to possibly correlate with repetitive RNA precursors.

STRUCTURAL analysis (Engineering); CRYSTALLIZATION

Crystals (2073-4352), 2017, Vol 7, Issue 12, p344

2073-4352

Academic Journal

10.3390/cryst7110344