Recombinant Human Laforin Expressed in Insect Cells: Expression, Purification, and Biochemical Characterizations

단신 (Notes)

EXPERIMENTAL METHODS

Cloning and Mutagenesis

The gene covering the full regions of human Laforin were amplified by polymerase chain reaction (PCR) and cloned into pHis (modified from Novagen’s pET32) vector using the BamHI/XhoI restriction enzyme sites. The resulting recombinant Laforin contains polyhistidine tags at its N- and Cterminus (MHHHHHHGSLVPRSENLYFQGS for N-terminus and LEHHHHHHHH for C-terminus). The phosphatase active site mutant (C266S) was obtained by applying the QuikChangeTM method. For the generation of baculovirus, wild type and mutant genes covering the Laforin and the C-terminal fused polyhistidine tag were amplified by PCR and cloned into pVL1393 baculovirus transfer vector (AB Vector) using BamHI/BglII restriction enzyme sites (Fig. 1).

Expression of Human Laforin in E. coli

The recombinant proteins were overexpressed in E. coli strain Rosetta 2(DE3) (Novagen). Transformed cells with the pHis-Laforin plasmid were grown in Terrific Broth to an OD600 of 0.5 at 37 ℃ and protein expression was induced by 0.1 mM isopropyl-D-thiogalactopyranoside at 18 ℃. Cells were further cultured at 18 ℃ for 48 h after protein induction, and were harvested by centrifugation at 10,000×g for 10 min.

Preparation of Recombinant Baculovirus and Expression of Human Laforin in Sf9 Cells

Spodoptera frugiperda (Sf9) cells were grown at 27 ℃ in Sf-900TM II SFM medium (Life Technologies) supplemented with 100 units/ml penicillin, 100 µg/ml streptomycin, and 1% fetal bovine serum (Life Technologies). The baculovirus transfer vectors pVL1393-Laforin and the linearized baculoviral genomic DNA vector (ProGreenTM, AB vector) were co-transfected into Sf9 cells using ProFectinTM reagent (AB vector). Recombinant baculoviruses generated by homologous recombination were harvested at 3 days post transfection and were amplified to produce high-titer virus stocks. The expression of Laforin was assessed at 2~3 days post infection by Western blot, using an anti-His tag antibody (Applied Biological Materials).

Purification of Recombinant Human Laforin

All of the protein purification steps were performed under ice-cold conditions. Cell pellets were resuspended and sonicated under hypotonic lysis buffer (20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) pH 7.4, 10 mM sodium chloride, and 1 mM ethylenediaminetetraacetic acid (EDTA), 10 mM 2-mercaptoethanol, 10% (v/v) glycerol, and 1 mM phenylmethylsulfonyl fluoride). The crude lysate was centrifuged at 40,000×g for 1 h at 4 ℃. The resulting supernatant was applied to an amylase column (New England BioLabs) and washed with wash buffer (20 mM HEPES pH 7.4, 150 mM sodium chloride, 1 mM EDTA, 10 mM 2-mercaptoethanol, 10% (v/v) glycerol, and 10 mM maltose). The recombinant Laforin was further eluted with elution buffer (20 mM HEPES pH 7.4, 150 mM sodium chloride, 1 mM EDTA, 10 mM 2-mercaptoethanol, 10% (v/v) glycerol, and 500 mM maltose). The eluted protein was pooled and concentrated prior to loading on to SuperdexTM 200 HiLoadTM 16/60 prep-grade column (GE Healthcare) equilibrated with the buffer of 20 mM HEPES pH 7.4, 300 mM sodium chloride, 1 mM EDTA, 10 mM 2-mercaptoethanol, and 500 mM maltose. The homogeneity of the purified recombinant Laforin was examined by SDS-PAGE with Coomassie Blue staining. The final protein concentration was determined by Bradford assay (Sigma).

Measurement of Dynamic Light Scattering

Dynamic light-scattering experiments were performed using a DynaPro-titan instrument (Wyatt technology). The data were measured at 25 ℃ at 0.125 mg/ml protein in the buffer of 100 mM Tris-HCl pH 8.5.

Phosphatase Activity Assay

pNPP, OMPF, and glycogen were used as substrates for measuring phosphatase activities of purified recombinant Laforins. The pNPP reaction mixture was 0.1 M sodium acetate, 0.05 M Bis-Tris pH 6.0, 2 mM dithiothreitol, and 50 mM pNPP. The OMFP mixture was 0.1 M Tris-HCl pH 8.0, 40 mM sodium chloride, 2 mM dithiothreitol, and 0.5 mM OMFP. 10 µg of recombinant Laforins were added to each reaction mixtures and stored at 30 ℃ for 20 min. The absorbance of the reaction products were measured at 405 nm for pNPP assay and at 477 nm for OMFP assay using a microplate reader (Molecular Devices). The phosphatase assay with glycogen was carried out under the reaction mixture of 100 mM sodium acetate, 50 mM Bis-Tris pH 6.0, 2 mM dithiothreitol, and 1 mg/ml glycogen at 37 ℃. The released phosphate was measured with malachite green reagent (Cayman Chemical) using the same microplate reader at 620 nm. Data were expressed as means ± standard deviation of at least three independent experiments. Statistical differences between groups were analyzed using one-way analysis of variance (ANOVA).