Background

Cyclin-dependent kinase-like 5 (CDKL5) is a serine/threonine protein kinase involved in the development of the human brain. Dozens of mutations of the CDKL5 gene are causative of CDKL5 Deficiency Disorder (CDD; OMIM 300203; 300672) [1, 2], a severe condition that is manifested with intellectual disability, autistic behavior, motor and visual impairments, infantile-onset refractory epilepsy, and many other symptoms [3]. Although no cure for CDD exists today, some studies proved that the restoration of CDKL5 activity through either protein or genetic intervention can revert CDD symptoms in mice and human models [4,49]

Measurement of pGFP fluorescence

After recombinant production, the equivalent of 1 OD cells was harvested and washed with PBS. Then, each sample was diluted with the same buffer to achieve the best signal-to-noise ratio in the fluorescence measurements that were carried out with a JASCO FP-750 spectrofluorometer at 25 °C (excitation at 488 nm and emission at 509 nm).

Analysis of the production of flCDKL5 and EB2 proteins

To analyze total protein productions, 1 OD pellets were solubilized in Laemmli Sample buffer and heated at 90 °C for 20 min. Then, total cellular extracts were resolved by SDS-PAGE and analyzed either by Coomassie staining or Western blot. For the solubility analysis, cells were lysed in 20 mM sodium phosphate buffer pH 7.0, supplemented with 0.5 M NaCl, 10% Glycerol, 0.1% Triton X-100, 20 U/mL DNAse I, 0.1 mg/mL lysozyme, 1 mM DTT and a protease inhibitor cocktail. Then, the soluble and insoluble fractions were segregated by centrifugation. Finally, the insoluble fraction was resuspended with lysis buffer in the same volume as the soluble fraction. 10 µg of soluble extract were analyzed by SDS-PAGE and the same volume of insoluble fraction was used as a control of solubility.

After SDS-PAGE runs, proteins were electroblotted to PVDF membranes using a semidry system. After the incubation with specific antibodies, the chemiluminescent signals were developed with the ECL method.

To detect flCDKL5, the membrane was blocked with PBS, 0.05% Triton X-100, 5% w/v milk for one hour. Then, CDKL5 (D-12): sc-376314 antibody (Santa Cruz Biotechnology) was diluted 1:1000 in the same buffer. After one hour of incubation at room temperature with the primary antibody, the membrane was washed with PBS, 0.05% v/v Triton X-100 three times (5 min each) and incubated with an anti-mouse antibody diluted 1:10,000 in PBS, 0.05% v/v Triton X-100, 5% w/v milk for one hour. Then, the membrane was washed again with PBS, 0.05% v/v Triton X-100 three times and the secondary antibody was detected using the ECL method.

For anti-FLAG Western blots, the membrane was blocked with PBS, 0.2% Tween 20, 5% w/v milk, for one hour. Then, Monoclonal ANTI-FLAG M2, Clone M2 (F1804, Sigma) was diluted 1:1000 in the same buffer. After overnight incubation at 4 °C with the primary antibody, the membrane was washed with PBS, 0.2% Tween 20 three times and incubated with an anti-mouse antibody diluted 1:5000 in PBS, 0.2% Tween 20, 5% w/v milk for one hour at room temperature. Then, the membrane was washed again with PBS, 0.2% Tween 20 three times and the secondary antibody was detected using the ECL method.

In the case of anti-His Western blots, the membrane was blocked with PBS, 5% w/v milk for one hour. Then, Monoclonal Anti-polyHistidine-Peroxidase clone HIS-1 antibody (A7058, Merck) was diluted 1:2000 in PBS, 0.05% Tween 20, 5% w/v milk. After one hour of incubation at room temperature with the antibody, the membrane was washed with PBS, 0.05% Tween 20 three times and it was developed.

To measure EB2 phosphorylation of Ser222, the membrane was blocked with TBST, 5% w/v milk for one hour. Then, anti-EB2 pS222 antibody (00117739, Covalab) was diluted 1:4000 in the same buffer. After overnight incubation at 4 °C with the primary antibody, the membrane was washed with TBST three times and incubated with an anti-rabbit antibody diluted 1:2000 in TBST and 5% w/v milk for one hour at room temperature. Then, the membrane was washed again with TBST three times and was developed.

Parallel Coomassie stained polyacrylamide gels were always used to ascertain that complex samples (i.e. total and soluble lysates) were correctly balanced in Western blot analyses.

Preparation of samples for N-terminal sequencing

To produce the catalytic domain of CDKL5 in E. coli, the primers named PhSumoCDKL5_NdeI_fw and PhCDKL5dC_XhoI_rv were used in a PCR on pB40-BCD2-107 (L). This gene encoding a Sumo-tagged version of CDKL5(1–352) was cloned into the pET40-b vector in frame with a C-terminal 8xHis tag using NdeI/XhoI double digestion. After recombinant expression in E. coli BL21(DE3), the recombinant cells were resuspended in 50 mM TrisHCl pH 8.0, 0.5 M NaCl, 20 mM imidazole and lysed by sonication in the presence of a protease inhibitor cocktail. After centrifugation (14,000 g, 4 °C, 60 min), the soluble fraction was loaded onto a HisTrap of 1 mL (Cytiva) and both the full-length protein, and its N-terminally truncated fragment were collected with a linear gradient of imidazole. A sample containing approximately 30 µg of the intact catalytic domain and 6 µg of the N-terminally truncated fragment were loaded onto SDS-PAGE and then electroblotted onto a PVDF membrane using 10 mM CAPS, 10% methanol pH 11.0 as the transfer buffer. The protein bands were made visible by Ponceau S staining and submitted to Edman sequencing at the Institute of Biosciences and Bioresources (CNR, Naples).

Enrichment of 107 (L) M10V from P. haloplanktis TAC125 lysate

Recombinant P. haloplanktis TAC125 was lysed with a chemical-enzymatic method. Briefly, the cell paste was resuspended in 20 mM sodium phosphate buffer pH 7.0, supplemented with 0.5 M NaCl, 10% glycerol, 0.1% Triton X-100, 20 U/mL DNAse I, 0.1 mg/mL lysozyme, 1 mM DTT, and a protease inhibitor cocktail to reach a final concentration of 14 OD/mL. After incubation at 4 °C for 20 min, the suspension was centrifugated (14,000 g for 45 min at 4 °C) to separate the soluble fraction from the cellular debris. Then, the soluble lysate was incubated with 0.15 mL of ANTI-FLAG M2 Affinity gel (Millipore) at 4 °C for 4 h and a gravity flow column chromatography was performed. The resin was washed with lysis buffer containing 1% Triton X-100, while the elution was performed using in 0.5 mL of the same buffer containing 175 µM 3xFLAG peptide.

EB2 purification

A codon optimized EB2 gene was synthesized by an external company and cloned into the pET40-b with NdeI/BamHI double digestion. The resulting gene encodes human EB2 with an N-terminal 6xHis tag (see the Additional file 1: Information for the nucleotide sequence). After recombinant expression, E. coli BL21(DE3) recombinant cells were lysed by sonication in 50 mM TrisHCl pH 8.0, 0.5 M NaCl, 5% glycerol, 20 mM imidazole supplemented with a protease inhibitor cocktail. The soluble fraction was recovered after a centrifugation (14,000 g for 45 min at 4 °C) and loaded onto a HisTrap of 1 mL (Cytiva). The target protein was eluted with 250 mM imidazole and loaded onto a a Hiload 16/600 Superdex 200 pg using 50 mM TrisHCl pH 8.0, 0.18 M NaCl as a running buffer for a final polishing step. The final preparation was stored at – 80 °C in 40 mM TrisHCl pH 8.0, 0.15 M NaCl, 1 mM DTT, 15% glycerol at 1.8 mg/mL protein concentration.

In vitro kinase assay

EB2 phosphorylation assays with enriched CDKL5 proteins were carried out using 200 nM EB2 and 100 nM of enzyme in 30 μL of 20 mM TrisHCl pH 7.7, 0.5 M NaCl, 10 mM MgCl2, 1 mM DTT, 0.7 mM ATP, complete protease inhibitor cocktail (Roche) and Halt phosphatase inhibitor cocktail (Thermo Fisher Scientific). The reactions were stopped after 30 min with 10 μL Laemmli Sample buffer 4 × and denatured at 70 °C for 20 min. 10 μL of each reaction were analyzed via either SDS-PAGE (for total CDKL5 and EB2 detection) or anti-EB2 pSer222 Western blot (for phosphorylated EB2 detection). As a negative control, a reaction was set up with flCDKL5 KD, a catalytically inactive CDKL5 variant. As a positive control, a reaction with commercial GST-CDKL5(1–498) (ab131695, abcam) was performed.

Statistics and reproducibility of results

The Data from the in cellulo kinase assays were statistically validated using the t-Student test comparing the mean measurements of experimental and control samples, both carried out as technical triplicates. The significance of differences between mean values was calculated using a two-tailed Student’s t-test. A p value of < 0.05 was considered significant.