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. Author manuscript; available in PMC: 2024 Nov 11.
Published in final edited form as: J Mol Biol. 2020 Oct 12;432(23):6028–6041. doi: 10.1016/j.jmb.2020.09.019

Fig. 2. Purification of SUN2-KASH complexes.

Fig. 2.

(a) Primary sequence and cartoon representation of overexpression constructs. For SUN2522-717 a cleavable trimerizing GCN4 tag was fused to the N-terminus, KASH contains a cleavable N-terminal 6x-Histidine-GB1 affinity/solubility tag. Arrowheads correspond to 3C cut sites, molecular weight of the tagged and digested constructs are denoted.

(b) SDS-PAGE analysis of purification steps of SUN2-KASH preparations. S, soluble protein fraction from bacterial lysate. E, Ni-NTA affinity elution fraction. P, pure protein complex after final gel filtration.

(c) Purification scheme as employed for all SUN2-KASH complex preparations in this study. 6x-His-GB1-KASH was Ni-affinity purified along with bound SUN2 coeluting. Ni-eluate was used for subsequent chromatographic purification steps. Within the chromatograms, regions highlighted in gray were pooled for subsequent purification or crystallization experiments.