Nanobody generation, expression, and purification.

Nanobodies were generated by the Core Facility Nanobodies, University Clinics Bonn, by repeated immunization of an alpaca with doubly T-loop phosphorylated Cdk7/Cyclin H/Mat1_230-309. After immunization, peripheral blood mononuclear cells (PBMCs) were isolated, the mRNA extracted and reverse-transcribed to cDNA. VHH sequences were amplified by PCR using specific primers and cloned into a phagemid vector for phage display. Cdk7/Cyclin H/Mat1-binding VHH were enriched by phage panning with biotinylated target complex and screened by ELISA.

Nanobodies for co-crystallization with Cdk7/Cyclin H/Mat1 were expressed in E. coli BL21 (DE3) cells from a modified pET28a vector. The expression construct contained an N-terminal pelB sequence for periplasmic translocation followed by a TEV protease-cleavable hexahistidine tag for purification (pelB-His6-TEV-NB). Nanobodies for biochemical studies were expressed in E. coli WK6 cells from a pHEN6 vector with N-terminal pelB sequence and C-terminal HA-His-tag (pelB-NB-HA-His6). Pre-cultures were prepared as described above and diluted into larger volumes of TB medium to an OD₆₀₀ of 0.1. Cultures were grown to OD₆₀₀ of 1.0 at 37°C for induction of expression. Protein expression was induced by adding IPTG to a final concentration of 1 mM, and the expression temperature was set to 30°C for 16 h. Bacteria were collected in 1-L buckets by centrifugation at 5000 rpm (JLA8.1 rotor, Beckman-Coulter) for 20 minutes. Bacterial pellets were subjected to periplasmic extraction of proteins or snap-frozen in liquid nitrogen and stored at −20°C for later use.

For purification, cells were resuspended in TES buffer (200 mM Tris pH 8.0, 0.65 mM EDTA, 500 mM sucrose) and incubated for 6 h at 4°C. Periplasmic extracts were generated by osmotic shock in 0.25x TES buffer for 16 h at 4°C. The extracts were cleared by centrifugation at 8000 rpm (JA25.50 rotor, Beckman-Coulter) for 45 min at 4°C, filtered through syringe filter with 0.45 μm pore size and subjected to affinity chromatography. The lysates were applied to HisTrap FF affinity columns (Cytiva) using an AKTA FPLC system (Cytiva). Columns were washed extensively with wash buffer (50 mM Tris pH 7.5, 150 mM NaCl, 10 mM imidazole) and eluted with wash buffer containing 500 mM imidazole. For crystallization, nanobodies were dialyzed against SEC buffer (20 mM HEPES pH 7.6, 150 mM NaCl) and the N-terminal His₆-tag was removed by TEV protease digestion. The proteins were further purified by SEC on a HiLoad 16/600 Superdex75 pg column (Cytiva) equilibrated with SEC buffer, followed by reverse Ni²⁺-NTA purification to remove non-cleaved protein. For biochemical assays, nanobodies were affinity-purified as described above and further purified by SEC on a HiLoad 16/600 Superdex75 pg column (Cytiva) equilibrated with SEC buffer (20 mM HEPES pH 7.6, 150 mM NaCl).