Cloning of four FST-type proteins into an expression vector and transformation of expression vectors
Since we previously used pMALc5x vector (New England Biolabs, MA, USA) in cloning for expression of bioactive chicken FST315 in an E. coli system (Lee et al. 2014), the same system was used in cloning four different FST-type proteins. The four FST-type proteins included FST288, NDFSD1/2, NDFSD1/1, and NDFSD1 (Fig. 1a). Gibson assembly cloning method (Gibson et al. 2009) was used to insert the DNA fragments of four FST-type proteins into pMALc5x vector separately. Gibson assembly primers for FST288 (GenBank Accession No. KT336491), NDFSD1/2 (GenBank Accession No. 336492), NDFSD1/1 (GenBank Accession No. 336493), and NDFSD1 (GenBank Accession No. 336494) fragments were synthesized (Fig. 1b), and inserts were prepared by PCR amplification using the Q5 High-Fidelity PCR kit (New England Biolabs) and chicken FST315 cDNA (Lee et al. 2014) as a template. PCR products were separated by agarose gel electrophoresis, and fragments were excised and purified before use in DNA assembly reaction with XmnI-linearized pMALc5x vector using Gibson Assembly® Cloning kit (New England Biolabs). Each assembly reaction contained approximately 100 ng of insert and 50 ng of the expression vector and incubated at 50 °C for 30 min following the manufacturer’s protocol. After the assembly reaction, the reaction mix was transformed into NEB 5-alpha competent E. coli strain (New England Biolabs). After an overnight growth at 37 °C, the pMAL-c5x plasmids containing respective inserts were extracted using a plasmid extraction miniprep kit (Promega) to confirm correct insertion by colony PCR.
Cytoplasmic expression of four FST-type proteins
Shuffle E. coli (New England Biolabs) were transformed with pMALc5x-FST288, pMALc5x-NDFSD1/2, pMALc5x-NDFSD1, and pMALc5x-NDFSD1/1 plasmids. After confirmation of correct insertion by colony PCR, 5 mL Luria–Bertani (LB) (1.2 % tryptone, 0.6 % yeast extract and 0.8 % NaCl) medium containing 100 μg/mL ampicillin and 0.2 % glucose were inoculated individually with the Shuffle E. coli harboring the inserts, and grown overnight at 30 °C with vigorous shaking. Then, 5 mL of the overnight cultures were transferred into 1 L fresh LB medium containing ampicillin in 2 L flask. When the culture reached to an optical density of 0.3–0.4 Å (600 nm) at 30 °C, the cultures were transferred to 4 °C for protein expression induced by adding Isopropyl β-d-1-thiogalactopyranoside (IPTG) to a final concentration of 0.4 mM under vigorous shaking. After induction for 8 days, E.coli pellet was harvested by centrifugation at 4000g for 10 min at 4 °C. Each gram (wet weight) of the cell pellet was resuspended in 5 mL of affinity column buffer (20 mM Tris–Hcl, 200 mM NaCl, 1 mM EDTA, pH 7.4) containing the Complete Mini Protease Inhibitor cocktail tablet (Roche, Mannheim, Germany). Two microliter of lysozyme (50 μg/mL) and 2 μL of DNase I (2500 units/ml) were added per 1 mL column buffer. The resuspended cell solution was lysed by sonication in short pulses of 15 s for 10 min in ice water bath. The soluble and insoluble fractions were prepared by centrifugation at 10,000g for 20 min at 4 °C. For each sample, the supernatants (soluble fraction) were collected, and the same volume of column buffer was used to resuspend the pellets (insoluble fraction). Total, soluble and pellet fractions were analyzed by SDS-PAGE to examine the presence of MBP-fused recombinant proteins (MBP-FST288, MBP-NDFSD1/2, MBP-NDFSD1, and MBP-NDFSD1/1).
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)
SDS-PAGE was performed with gels containing l2.5 % polyacrylamide and 0.1 % SDS following the procedure of Laemmli (1970). Samples were mixed with 3X loading buffer which were under reducing conditions. Before loading the sample onto the SDS-PAGE gel, samples were boiled at 100 °C for 5 min.
Amylase affinity purification of MBP-fused FST-type proteins
Supernatant cell extracts were diluted with affinity column buffer in a 1:5 ratio, and filtered through 0.45 μm filter, then was loaded into an amylose resin column equilibrated with 100 mL of column buffer. After loading, the pass-through was collected at a rate of 0.5 ml/min and washed with 100 mL of column buffer. Proteins bound to the column were then eluted with elution buffer (column buffer with 10 mM maltose) at a flow rate of 0.5 ml/min. 5 mL fractions were collected during elution, and the absorbance was monitored at 280 nm. After SDS-PAGE analysis of the presence of recombinant protein in fractions, fractions containing recombinant proteins were pooled.
Heparin affinity purification of MBP-fused FST-type proteins
For further purification of amylose affinity-purified MBP-fused proteins, the pooled elutions were subjected to heparin affinity column (Bio-Rad, CA, USA) previously equilibrated with column buffer. The pass-through was collected at a rate of 1 ml/min. The column was then washed with 100 mL of column buffer. Proteins bound to the column were then eluted with elution buffer (column buffer with 1 M NaCl). After SDS-PAGE analysis of the presence of recombinant protein in fractions, fractions containing recombinant proteins were pooled, followed by dialysis in phosphate buffered saline solution.
Affinity-purified MBP-fused proteins were subjected to a 12.5 % SDS-PAGE, followed by a transfer onto a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked for 1 h at room temperature with 5 % non-fat dry milk in Tris-buffered saline with 0.1 % Tween-20 (TTBS). The membranes was incubated with the goat anti-FST (1:5,000 in TTBS, R&D Systems, MN, USA) overnight at 4 °C. The membrane was then washed three times (10 min for each wash) with TTBS, followed by incubation with 1:10,000 alkaline phosphatase-conjugated anti-goat IgG (Sigma, MO, USA) in TTBS for 1 h. After washing, the membrane was developed using BCIP/NBT substrate (nitrobluetetrazolium and bromo-chloro-indolyl phosphate from Sigma).
Bioactivity test of MBP-fused FST-type proteins by pGL3-(CAGA)12 Luc-luciferase reporter system
The capacities of MBP-FST288, MBP-NDFSD1/2, MBP-NDFSD1, and MBP-NDFSD1/1 to suppress the bioactivity of three FST-binding proteins (MSTN, GDF11, and activin A, all from R&D Systems) were examined by a procedure that was used in examining the bioactivity of MBP-fused chicken FST315 (Lee et al. 2014). Briefly, cells stably transformed with pGL3-(CAGA)12 luciferase reporter construct (Cash et al. 2012) were seeded in a 96 well plate for 24 h at 37 °C with 5 % CO2. The medium was replaced with 100 μL serum-free DMEM containing 1 nM MSTN, GDF11 or activin A plus various concentrations of FST-type proteins, then incubated for 24 h. After removing the medium, Bright-Glo luminescence substrate (Promega) was added, and luminescence was measured. The % inhibition of MSTN, GDF11 or activin A activity was calculated by the following formula: % inhibition = (luminescence at 1 nM MSTN, GDF11 or activin A-luminescence at each ligand concentration) × 100/(luminescence at 1 nM MSTN, GDF11 or activin A-luminescence at 0 nM MSTN, GDF11 or activin A). The MSTN-, GDF11- or activin A-inhibitory activity was analyzed by regression analysis using Prism 5 program (Graphpad, CA, USA). To examine the differences in MSTN-, GDF11- or activin A-inhibitory capacity of these proteins, IC50 (ligand concentration inhibiting 50 % of MSTN, GDF11 or activin A activity) values were estimated using a non-linear regression model defining dose response curve. The equation for the model was as follows: Y: Bottom + (Top − Bottom)/[1 + 10^(X − LoglC50)], where Y is % inhibition, Bottom is the lowest value of % inhibition, Top is the highest value of % inhibition, and X is Log ligand concentration IC50 values were analyzed by ANOVA (Analysis of Variance) using the same program.