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. 2016;569:63-78.
doi: 10.1016/bs.mie.2015.08.011. Epub 2015 Sep 6.

Purification and Structural Analysis of SUN and KASH Domain Proteins

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Free PMC article

Purification and Structural Analysis of SUN and KASH Domain Proteins

F Esra Demircioglu et al. Methods Enzymol. .
Free PMC article

Abstract

Molecular tethers span the nuclear envelope to mechanically connect the cytoskeleton and nucleoskeleton. These bridge-like tethers, termed linkers of nucleoskeleton and cytoskeleton (LINC) complexes, consist of SUN proteins at the inner nuclear membrane and KASH proteins at the outer nuclear membrane. LINC complexes are central to a variety of cell activities including nuclear positioning and mechanotransduction, and LINC-related abnormalities are associated with a spectrum of tissue-specific diseases, termed laminopathies or envelopathies. Protocols used to study the biochemical and structural characteristics of core elements of SUN-KASH complexes are described here to facilitate further studies in this new field of cell biology.

Keywords: KASH peptide; LINC complex; Nesprin; Nuclear envelope; SUN domain; SUN protein.

Figures

Figure 1
Figure 1
Schematic drawing of the expression constructs for human SUN2 (A), and KASH (B). Each SUN2 fragment includes the SUN domain (residues 540-717) and preceding lumenal segments of different length, predicted to form coiled-coils (CC). The SUN2 (residue 335-717) fragment is N-terminally fused to a 3C-cleavable 6xHis tag, whereas a 6xHis:triGCN4 tag is used for a shorter SUN2 fragment (residues 522-717). KASH motifs are C-terminally attached to 3C-cleavable MBP, superfolder GFP (sfGFP) or GB1 tags. Short flexible linkers are incorporated around the 3C cleavage sites to enhance removal of the fusion tags.
Figure 2
Figure 2
Purification schemes for SUN2-KASH complexes. (A) Purification of the SUN2-KASH3 complex is illustrated to exemplify the strategy used for SUN2 / MBP-KASH constructs. A representative gel filtration elution profile at each step is shown, and the proteins eluted under each peak are indicated. (B) Purification of the SUN2-KASH5 complex is illustrated to demonstrate the strategy used for SUN2 / GB1-KASH constructs.
Figure 3
Figure 3
In vitro SUN-KASH binding experiments. SUN-KASH binding was analyzed by gel filtration on a Superdex S200 HR10/300 column. Each panel shows a representative gel filtration profile and corresponding SDS-PAGE as follows: (A) Apo-SUN2 in buffer 1. (B) Apo-SUN2 in buffer 2. (C) Apo-SUN2 and sfGFP-KASH2 in buffer 1. (D) Apo-SUN2 and sfGFP-KASH2 in buffer 2. Asterisks are used to mark each peak, denoting the SUN2 monomer peak as S1, the SUN2 trimer as S3, the SUN2 trimer bound to sfGFP-KASH2 as S3K, and unbound sfGFP-KASH2 as K. Solid lines on each chromatogram represent the 280 nm trace; dashed lines represent the 488 nm trace. Buffer 1 contains 20 mM HEPES pH 8.0 and 100 mM KCl. Buffer 2 contains 10 mM Tris/HCl pH 8.0, 150 mM NaCl and 1 mM KCl. Each Coomassie-stained SDS-PAGE gel shows 1 ml fractions covering the 7–18 ml elution segment. SUN oligomerization is buffer-dependent. Buffers that enable SUN trimerization are required for SUN-KASH binding experiments.

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