Fig 1.
From designing the gene construct to the expression and purification of αB-lir hybrid protein.
A) pET28b(+) vectors containing the hybrid gene (αB-lir) are demonstrated. The positions of NcoI and XhoI restriction sites are also indicated. The translation frame on this vector shows the amino acid sequence of αB-lir hybrid protein. The human αB-Cry gene is portrayed in purple, and the primary structure of the LPP is shown as red. Also, M stands for the methionine residue that provides a CNBr specific cleavage site. In the primary structure of the human αB-Cry gene, methionine 68 and proline 130 are substituted with isoleucine and valine residues, respectively. B) The expression of αB-lir hybrid protein was assessed by SDS-PAGE analysis (gel 12%). The αB-lir expression in the absence and presence of 0.25 mM IPTG is indicated, and M shows the protein mass markers. C) The αB-lir was purified using the precipitation method followed by a DEAE column. Then, the pure αB-lir was analyzed on a reducing SDS-PAGE (gel 12%). Lanes 1 and 2 stand for human αB-Cry and αB-lir hybrid protein, respectively. Also, M indicated the protein mass marker.
Fig 2.
From the specific chemical cleavage of the αB-lir hybrid protein to the purification of LPP.
A) The CNBr was used for cleaving a specific peptide bond at the boundary methionine between partner protein (human αB-Cry) and LPP. Lanes 1 and 2, respectively, indicate human αB-Cry and αB-lir, while lane 3 shows αB-lir after the specific chemical cleavage at the boundary methionine. M is the molecular mass marker. B) The αB-lir after the chemical cleavage was subjected to a Sephadex G50 gel filtration column. The upper and lower panels show the first and the second round of purification. The pooled fractions rich in the LPP were analyzed by SDS-PAGE (gel 18%). Lane 1 in the upper and lower panels respectively indicates a semi-purified and a highly pure sample of the LPP. C) The pure fraction of LPP was then subjected to a reverse-phase (ProntoSIL 200-5-C18, 250 × 4.6 mm) high-performance liquid chromatography column (HPLC) equipped with a UV detector (Smartline 2500 KNAUER). The sample was eluted at a 1 mL/min flow rate with a 0–60% linear gradient of acetonitrile over 15 min at 25°C.
Fig 3.
Mass spectroscopy analyses of the LPP.
MALDI-TOF mass spectroscopy analysis of the LPP (A), and Orbitrap High-resolution Liquid chromatography mass spectrometer analysis of m/z of the peptide (B).
Fig 4.
Raman spectral analyses of the αB-lir hybrid protein and LPP in the fingerprint region.
The structure of αB-lir and LPP was characterized by Raman spectroscopy in the fingerprint region. Raman spectra of the LPP (A) and αB-lir hybrid protein (B) were indicated.
Fig 5.
The secondary structure analyses of LPP and αB-lir.
For the analyses of the secondary structures, three different methods as Raman (A), ATR-FTIR (B) and far UV-CD (C) were applied. During the Raman and FTIR assessments, the samples were used as powder while the amide band I was deconvoluted by GRAMS (version 9.2) [38]. In the far UV-CD analyses, the LPP was dissolved in phosphate buffer pH 8.1, while αB-lir and αB-Cry were prepared in 100 mM acetate buffer, pH 5.2. In the far UV-CD assessments, the protein/peptide concentration was fixed at 0.2 mg/mL, and the data were deconvoluted by DichroWeb [43]. The upper, middle and lower panels respectively stand for the secondary structure analyses of the LPP, αB-lir and αB-Cry.
Table 1.
The protein secondary structure elements (%) of LPP, αB-lir and αB-Cry obtained by three different methods as Raman, ATR-FTIR and far UV-CD.
Fig 6.
Oligomerization status and surface hydrophobicity of αB-lir hybrid protein.
A) The hydrodynamic diameters of αB-lir hybrid protein and human αB-Cry (1 mg/mL in phosphate buffer, pH 7.4) were measured at different temperatures by a DLS instrument. B) For the ANS fluorescence assessments, the αB-lir and αB-Cry were prepared in 50 mM sodium phosphate buffer, pH 7.4 and the protein concentrations were fixed at 0.15 mg/mL. Then, their surface hydrophobicity analyses were performed at various temperatures in the presence of a fixed ANS concentration (100 μM). The excitation of the protein/ANS complex was done at 365 nm, and the emission spectra were collected between 400–600 nm. Also, the excitation and emission slits were fixed at 5 and 10 nm, respectively [44].
Fig 7.
The glucose-lowering activity of LPP and αB-lir.
The glucose-lowering effects (IPGTT) of LPP (A) and αB-lir (B) were studied. A single-dose of the LPP (200 μg/kg body weight) and the αB-lir hybrid protein (1.4 mg/kg body weight) was injected to the mice and the blood glucose concentration was measured during 120 minutes [46]. The concentration of injected glucose was 1.5 mg/g body weight of the normal mice, and the injection was done at time zero. In the case of αB-lir, the blood glucose level was monitored after 24 hours of the initial injection. The blood glucose levels of STZ-induced diabetic mice (n = 6) were also measured after a single-dose, 200 μg/kg and 1.4 mg/kg body weight, respectively, for the injection of the LPP (C) and αB-lir (D). The data were significantly different from the control group (p<0.05).
Fig 8.
The analyses of the insulin induced secretion of LPP and αB-lir.
The effect of LPP (200 μg/kg body weight) (A) and αB-lir (1.4 mg/kg body weight) (B) on stimulation of the insulin secretion was studied by a single-dose injection. The plasma insulin levels of normal mice (n = 6) were measured by an appropriate insulin ELISA kit [51]. In the case of αB-lir, the plasma insulin level was monitored after the initial injection. The glucose concentration was 1.5 mg/g body weight of normal mice as injected at the time zero. All data were significantly different from the control group (p<0.05).