Recombinant protein cloning, expression, and purification

Fragments of Cnn-C-N and Cnn-T-N used in co-IP experiments were amplified from the pDONR-Cnn-C and pDONR-Cnn-T vectors described above by PCR and inserted into a pDEST-HisMBP (#11085; Addgene) vector by Gateway cloning (Thermo Fisher Scientific). Proteins were expressed in E. coli (BL21-DE3) and purified using affinity chromatography. MBP-tagged fragments were purified by gravity flow through amylose resin (New England Biolabs) and step elution in maltose. The concentration of each fraction was determined on a Nanodrop and peak fractions were diluted 1:1 with glycerol and stored at −20°C. Truncated fragments of Cnn-C were made by modification of the pDONR-Cnn-C-N entry clone. The N-terminal region was removed by a Quikchange reaction (Agilent Technologies), and the resulting shortened fragment was inserted into the pDEST-HisMBP destination vector via a Gateway reaction.

Phosphomimetic fragments were created by modifying the pDONR-Cnn-C-N entry clone. The pDONR-Cnn-C-N backbone was linearized by PCR or by digestion, omitting the phospho-patch to be replaced. Phosphomimetic patches in which all S/T residues were swapped for D/E residues, respectively, were synthesized either by PCR using two overlapping primers or by GENEWIZ. They were inserted into the linear backbone by HiFi assembly (New England Biolabs). Entry clones were checked by restriction enzyme digest and sequencing before being inserted into the pDEST-HisMBP destination vector via a Gateway reaction.

pRNA vectors were made by modification of the pDONR-Cnn-C-PReM^P vector containing phosphomimetic mutations in the PReM domain (Conduit et al., 2014a). N-terminal variants were introduced by restriction digests (SspI-HF and AatII) of pDONR-Cnn-C, pDONR-Cnn-T, and pDONR-Cnn-C-PReM^P entry clones. Fragments were combined as necessary by HiFi assembly to create new pDONR vectors that were inserted into a pRNA-GFP or pRNA-mKate destination vector (Conduit et al., 2014a) via a Gateway reaction. The Cnn-T-N fragment was inserted directly into pRNA-GFP destination vectors via Gateway cloning.

Fragments of CDK5RAP2 were synthesized by GENEWIZ, amplified by PCR, and cloned into a pCMV-GFP vector (gift from Jens Lüders, Institute for Research in Biomedicine [IRB Barcelona], The Barcelona Institute of Science and Technology, Barcelona, Spain) by restriction digest and HiFi assembly (New England Biolabs).

Primers used are listed in Table 1.