The reference sequence of the chicken ApN gene was obtained from GenBank, under the reference number NM_206991.1. The full-length sequence of the chicken ApN gene is shown in Figure 1. The coding region of the ApN gene for its insertion into the vector pET32a was amplified by PCR using the oligonucleotides (forward: 5′-GACGACGACGACAAGGCCATGAG GGGCTCAGTAGGCTTC-3′; reverse: 5′-GCTCGAGTGCGGCCGCAA GCTTGACGGTCATCTGTGTCTGGGTA-3′), which introduced a HindIII (Cat. #1060A, Takara Bio, Shiga, Japan) site at the 3′ end. The amplified ApN gene fragment was inserted into the vector pET32a using the In-Fusion HD enzyme (Cat. #638909, Takara Bio, Shiga, Japan), resulting in the plasmid named pET32a-adiponectin. All of the constructed plasmids were transformed into E. coli DH5α and confirmed by PCR and DNA sequencing.

Figure 1 The chicken adiponectin cDNA contains 735 bp, which translates into a protein that is 244 amino acids in length.

Competent cells were removed from −80 °C and thawed on ice, then 5 µL of the plasmid DNA and 50 µL of the competent cells were mixed gently by pipetting up and down. The mixture was placed on ice for 30 min and then transferred to a 37 °C water bath for 45 s, then put back on ice for 2 min. Twenty microliters of SOC medium were added to the bacteria and incubated in a shaking incubator at 37 °C for approximately 60 min. The cells were spread onto LB agar plates with a selection antibiotic by using a sterile spreader, and then incubated at 37 °C for 6 h.

Plasmid DNA purification with the TOOLS Plasmid Mini kit (Cat. #TT-A03-3, Biotools, New Taipei City, Taiwan) is based on the alkaline lysis method, followed by the adsorption of DNA onto a silica membrane. The bacterial cells were harvested in a microcentrifuge tube by centrifugation at 12,000 rpm (~13,400×g) for 1 min at room temperature, and then all traces of supernatant were removed. Pelleted bacterial cells were resuspended in 200 μL of buffer P1. Two hundred microliters of buffer P2 were added and mixed thoroughly by inverting the tube gently 10 times, then 300 μL of buffer P3 were added and mixed thoroughly by inverting the tube 10 times. The solution, which became cloudy, was centrifuged at 12,000 rpm (~13,400×g) for 10 min, and a compact white pellet formed. The supernatants were applied to Spin Column CP3 by decanting or pipetting, and then centrifuged at 12,000 rpm (~13,400×g) for 1 min, then the flow-through was discarded. Spin Column CP3 was washed by adding 400 µL of Buffer PD and centrifuging at 12,000 rpm (~13,400×g) for 1 min, and the flow-through was discarded. Spin Column CP3 was washed by adding 700 µL of Buffer PW and centrifuging at 12,000 rpm (~13,400×g) for 1 min, and the flow-through was discarded. To remove the residual wash buffer PW, Spin Column CP3 was centrifuged at 12,000 rpm (~13,400×g) for an additional 3 min. Spin Column CP3 was placed into a clean 1.5 mL microcentrifuge tube. To elute the DNA, 50–100 µL of Buffer EB or water (pH 7.0–8.5) were added to the center of each spin column (CP3), stood for 2–5 min, and centrifuged at 12,000 rpm (~13,400×g) for 2 min. The collected DNA was stored at 4 or −20 °C.

The presence of the gene insertion was verified using DNA sequencing, and then the plasmid was transformed into E. coli BL21 (Cat. #69450-3CN, Novagen, Madison, USA). A single colony was selected for the inoculation of 200 mL of LB medium, which contained 100 µg/mL ampicillin. For induction, isopropyl-β-D-thiogalactoside (IPTG, Cat. #N714-10ML, Amresco LLC, Solon, USA) was added to the culture to a final concentration of 1 mM, and simultaneously supplemented with 100 µg/mL ampicillin (Cat. #A9518, Sigma, St. Louis, USA). The culture was further incubated at 30 °C with shaking for an additional 3 h. The culture medium was centrifuged at 5000 rpm for 20 min, and the induced bacterial pellet was harvested and stored at −20 °C. For recombinant protein purification, the bacterial pellet was suspended in 20 mL crude extract buffer (50 mM Tris–HCl, 100 mM NaCl, 10 mM imidazole, and 1 µM phenylmethysulfonyl fluoride, pH 7.5), and then broken up using JY88-IIN sonication (Scientz, Ningbo, China). The supernatant fraction with soluble protein from the lysate was collected via centrifugation at 10,000 rpm for 20 min. The supernatant was loaded onto 0.5 mL Ni-NTA His-Tag Purification Resin (Cyrusbioscience, New Taipei City, Taiwan) that had been pre-equilibrated with lysis buffer (50 mM Tris-HCl and 100 mM NaCl, pH 7.5) containing 10 mM imidazole, and incubated on ice at 300 Mot 1/min for 1 h to enable optimal binding between the fusion protein and the resin. The mix of supernatant and resin was poured into Poly-Prep^® prepacked columns (Cat. #731-1550, Bio-Rad, Hercules, USA). After washing the column with 1 mL lysis buffer containing 10 mM imidazole to remove the unbound protein, the bounded protein was eluted with increasing concentrations of imidazole (50, 125, 250, and 500 mM) in 1.5 mL of lysis buffer, and the fractions were collected and kept at 4 °C.

Eight microliters of the 4× reducing sample buffer (0.25 M Tris-HCl, 8% SDS, 30% glycerol, 0.02% Bromophenol Blue, and 0.3 M DTT, pH 6.8) was added to 24 µL of protein samples (a total volume of 32 µL), then boiled under reducing conditions in the 1.5-mm-thick gel, which was cast with a 12.5% separating and a 4% stacking gel mixture, and loaded with 2 µL Precision Plus Protein^TM Dual Color Standards (Cat. #161-0374, Bio-Rad, Hercules, USA) and 30 μL aliquots of each sample. The electrophoresis was performed in a Mini-PROTEAN Tetra Cell (Cat. #1658006FC, Bio-Rad, Hercules, USA) filled with appropriately 1× running buffer at 100 V for 30 min, and then at 180 V for approximately 1 h, until the blue line almost reached the bottom of the gel. To visualize the protein bands and determine the purity and molecular weight of the protein, the gels were stained with Coomassie Brilliant Blue R‐250 (Cat. #CO-3360, W. S. Simpson, Hertfordshire, England) and shaken at room temperature for 30–60 min until the bands were visible. The staining solution was then decanted at room temperature on a shaker until the desired background was achieved.

The chicken hepatocellular carcinoma cell line, LMH (CRL-2117, ATCC) was cultured in Waymouth’s MB 752/1 medium (Biological Industries, Cromwell, CT, USA) with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA) and 1% 100 units/mL penicillin, 100 mg/mL streptomycin, and 0.025 mg/mL amphotericin B (Biological Industries, Kibbutz Beit Haemek, Israel). LMH cells were incubated in 6-well plates at an initial density of 5×10⁵/well, in a 5% CO₂ humidified atmosphere incubator at 37 °C. When the cells reached 95% confluence, they were starved with serum-free Waymouth’s MB 752/1 medium for 8 h. For the fatty acid treatment, PA (Cayman Chemical Company, Ann Arbor, MI, USA) and OA (Cayman Chemical Company, Ann Arbor, MI, USA) were both dissolved in ethanol (Sigma-Aldrich Corporation, St. Louis, MO, USA), and the dissolved PA or OA were aliquoted and stored at −20 °C. The 200 μM PA and 200 μM OA were conjugated with 1% BSA with low fatty acid, low endotoxin, and low IgG (US Biological Life Sciences, Swampscott, MA, USA) in complete Waymouth’s MB 752/1 medium, with sonication for at least 30 min prior to the treatment of the cells. The BSA-conjugated OA and PA were added to the wells for 48 h, with or without recombinant ApN.

Total RNA from the LMH cells was extracted using GENEzol^TM Reagent (Geneaid Biotech, Ltd., New Taipei City, Taiwan). The RNA concentration was measured at 260/280 nm using a NanoDrop^TM One (Thermo Fisher Scientific, Waltham, MA, USA). For each reverse-transcribed sample, 7 μg RNA were treated with TURBO^TM DNase (Thermo Fisher Scientific, Waltham, MA, USA) to remove the genomic DNA. The DNase-treated total RNA was used to generate cDNA using a High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific, Waltham, MA, USA). Quantitative real-time PCR reactions were performed via a StepOnePlus^TM Real-Time PCR System (Applied Biosystems, Foster City, CA, USA), with a DyNAmo Flash SYBR Green High-ROX Detection Kit (Finnzymes, Espoo, Finland). The conditions of the program were as follows: 2 min at 95 °C for polymerase activation, 40 cycles at 95 °C for 5 s for denaturation, and 60 °C for 30 s for annealing/extension. The specific primer sequences are listed in Table 1.

Table 1

Gene	Forward primer	Reverse primer
β-actin	5′-gtgatggactctggtgatgg-3′	5′-ccatgtgtcctggaaatcct-3′
SREBP1c	5′-gccctctgtgcctttgtcttc-3′	5′-actcagccatgatgcttcttc -3′
FAS	5′-aagcaattcgtcacggacag-3′	5′-ggcaccatcaggactaagca-3′
ACC	5′-agtcctgattgagcatggca-3′	5′-ctccagatggcggtagattc -3′
CPT1α	5′-ctgggttattgccacgaagc -3′	5′-gccatggctaaggttttcgt -3′
PPARα	5′-acggagttccaatcgc-3′	5′-aacccttacaaccttcacaa -3′

M - protein markers; CE - crude extract cell lysate; SP - soluble protein; UN - unbound protein.

Statistical analyses were performed using GraphPad software (version 5 for Windows). The collected data were tested via one-way ANOVA, and the mean differences were compared using Tukey’s multiple comparison test. Significance was declared at P≤0.05.

To construct the combination of an optimized expression vector with bacterial expression systems that were adapted for high-yield recombinant protein expression, the ApN coding sequence was cloned into the pET32a vector using the NcoI and HindIII restriction sites. The antibiotic resistance gene-selected vectors were transformed into competent DH5α cells for storage and scale-up applications. Colony PCR reactions were performed to screen the clones, and the PCR products with the correct product sizes were sequenced for verification. All of the sequence alignments of the cloned ApN fusions displayed 100% similarity with the sequence of chicken ApN published in GenBank.

The E. coli host strain BL21, transformed with the expression vector pET32a-adiponectin, produced a recombinant fusion protein of approximately 45 kDa after IPTG induction. Trx-adiponectin containing a (His)₆-tag between the fusion partners Trx-tag and ApN was purified using Ni-NTA His-Tag Purification Resin. The recombinant protein was eluted with four lots of 1.5 mL volumes of buffer containing 50, 125, 250, and 500 mM imidazole. The purified recombinant protein was collected, and its purity was assessed using SDS-PAGE. Most of the recombinant protein was detected at the 125 mM imidazole buffer-eluted fraction (Figure 2A), and approximately 95% of the protein consisted of the soluble fusion protein Trx-adiponectin, with a molecular mass of approximately 45 kDa, which was consistent with the size of the predicted Trx-adiponectin fusion protein. SDS-PAGE under non-reducing conditions was performed for the detection of the oligomerization process in ApN. The majority of the Trx-adiponectin fusion protein migrated as monomers at approximately 45 kDa, and as dimers of approximately 90 kDa, with a few trimers being present at approximately 135 kDa (Figure 2B). Approximately 1.5 mg of pure Trx-adiponectin was obtained from bacterial cultures, with a 1.24 g pellet, and the yield accounted for 5% of the total bacterial protein.

Figure 2 A) Expression of chicken recombinant adiponectin from pET32a-adiponectin in E. coli. Samples were eluted using buffers containing 50 mM imidazole (lane 1), 125 mM imidazole (lane 2), 250 mM imidazole (lane 3), and 500 mM imidazole (lane 4). B) The expression profile of recombinant adiponectin via non-reducing SDS-PAGE. Samples were eluted by buffers containing 125 mM imidazole (lane 1).

Compared with the untreated cells, OA and PA treatment increased lipid accumulation in the LMH cells. Additionally, the treatment of chicken recombinant ApN (10 μg/mL and 20 μg/mL) could ameliorate lipid accumulation in the LMH cells (Figure 3A). The quantification data from Oil Red O staining showed the same pattern as the representative figure (Figure 3B).

Figure 3 A) Representative images of Oil Red O staining; B) Quantification of Oil Red O staining. Con - control; OAPA - oleic acid and palmitic acid; Ad10 - chicken recombinant adiponectin, 10 μg/mL; Ad20 - chicken recombinant adiponectin, 20 μg/mL. Data were expressed as relative mean ± SEM (n = 5 in each group). Different letters indicate statistical differences.

The OA and PA challenges enhanced lipogenic genes such as SREBP1c, FAS, and ACC in LMH cells (Figure 4A, 4B, and 4C). Additionally, CPT1-α and PPARα, which represented lipolysis-related genes, decreased with OA and PA treatment (Figure 4D and 4E). However, chicken recombinant ApN can retard the lipid accumulation caused by OA and PA treatment, through an increase in β-oxidation, and a decrease in adipogenesis.

Figure 4 A) SREBP1c; B) FAS; C) ACC; D) CPT1α; E) PPARα. Con - control; OAPA - oleic acid and palmitic acid; Ad10 - chicken recombinant adiponectin, 10 μg/mL; Ad20 - chicken recombinant adiponectin, 20 μg/mL. SREBP1c - sterol regulatory element-binding transcription factor 1c; FAS - fatty acid synthase; ACC - acetyl-CoA carboxylase; CPT1α - carnitine palmitoyltransferase 1α; PPARα - peroxisome proliferator-activated receptor α. The mRNA were determined using real-time PCR and normalized to β-actin. Data were expressed as the relative mean ± SEM (n = 5 in each group). Different letters indicate statistical differences.