Journal: Frontiers in Pharmacology

Article Title: Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

doi: 10.3389/fphar.2023.1279516

Figure Lengend Snippet: Recombinant plasmid construction and expression of the hbFGF protein. (A) Schematic diagram of the mpET3c-hbFGF recombinant plasmid construction. (B) Kanamycin resistance detection of the mpET3c-hbFGF recombinant plasmid. 1, E. coli BL21 (DE3) plysS-pET3c strain. 2, E. coli BL21 (DE3) plysS-mpET3c/hbFGF engineered strain. (C) Restriction enzyme analysis of the mpET3c-hbFGF recombinant plasmid. Lanes 1 and 4, DNA molecular weight marker. Lane 2, plasmid before digestion. Lane 3, plasmid after digestion. (D) SDS-PAGE (left) and Western blot (right) analyses of the expressed hbFGF protein in E. coli BL21(DE3) plysS. Lanes 1 and 4, non-induced. Lanes 3 and 5, induced by IPTG for 4 h. Lane M, molecular weight marker. Black arrows indicate hbFGF.

Article Snippet: The polyclonal rabbit anti-human bFGF antibody (Cat.No: 3196S) was acquired from Cell Signaling Technology (United States), while the secondary antibody (goat anti-rabbit lgG/HRP antibody, Cat.No: HS101-01) was procured from Transgene Biotech (Beijing, China).

Techniques: Recombinant, Plasmid Preparation, Expressing, Molecular Weight, Marker, SDS Page, Western Blot

Journal: Frontiers in Pharmacology

Article Title: Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

doi: 10.3389/fphar.2023.1279516

Figure Lengend Snippet: Tested cultural conditions for hbFGF production on a flask scale.

Article Snippet: The polyclonal rabbit anti-human bFGF antibody (Cat.No: 3196S) was acquired from Cell Signaling Technology (United States), while the secondary antibody (goat anti-rabbit lgG/HRP antibody, Cat.No: HS101-01) was procured from Transgene Biotech (Beijing, China).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

doi: 10.3389/fphar.2023.1279516

Figure Lengend Snippet: Optimizing of fermentation parameters of hbFGF E. coli strain in 30 mL of LB medium. (A) The 12-h growth curve of the hbFGF E. coli strain in 30 mL of LB medium at 37°C and 200 rpm. (B–E) The 12-h growth curve of hbFGF E. coli strain under different conditions, including (B) glucose concentrations in the range of 0.5–20 g/L, (C) pH in the range of 6.6–7.4, (D) temperatures of 32°C, 34°C, 36°C, and 38°C, and (E) medium volume (30, 50, 75, and 100 mL). (F) The expression level of hbFGF (f1) and bacterial density (f2) after induction at different OD 600 values with 0.8 mM IPTG for 1–6 h. (G) The 10-h growth curve of the hbFGF E. coli strain under different inoculations. All experiments were performed in a 250-mL shake flask. Asterisks indicate significant difference (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 3).

Article Snippet: The polyclonal rabbit anti-human bFGF antibody (Cat.No: 3196S) was acquired from Cell Signaling Technology (United States), while the secondary antibody (goat anti-rabbit lgG/HRP antibody, Cat.No: HS101-01) was procured from Transgene Biotech (Beijing, China).

Techniques: Expressing

Journal: Frontiers in Pharmacology

Article Title: Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

doi: 10.3389/fphar.2023.1279516

Figure Lengend Snippet: Three-dimensional response surface for the bacterial density and hbFGF expression level. (A) Effects on the bacterial density of (a1) temperature and pH; (a2) temperature and IPTG concentration; (a3) temperature and NH 4 Cl concentration; (a4) temperature and induction time; (a5) pH and IPTG concentration; (a6) pH and NH 4 Cl concentration; (a7) pH and induction time; (a8) IPTG concentration and NH 4 Cl concentration; (a9) IPTG concentration and induction time; and (a10) NH 4 Cl concentration and induction time. (B) Effect on the hbFGF expression level of (b1) temperature and pH; (b2) temperature and IPTG concentration; (b3) temperature and NH 4 Cl concentration; and (b4) temperature and induction time.

Article Snippet: The polyclonal rabbit anti-human bFGF antibody (Cat.No: 3196S) was acquired from Cell Signaling Technology (United States), while the secondary antibody (goat anti-rabbit lgG/HRP antibody, Cat.No: HS101-01) was procured from Transgene Biotech (Beijing, China).

Techniques: Expressing, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

doi: 10.3389/fphar.2023.1279516

Figure Lengend Snippet: The optimal induction conditions for the hbFGF fermentation.

Article Snippet: The polyclonal rabbit anti-human bFGF antibody (Cat.No: 3196S) was acquired from Cell Signaling Technology (United States), while the secondary antibody (goat anti-rabbit lgG/HRP antibody, Cat.No: HS101-01) was procured from Transgene Biotech (Beijing, China).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

doi: 10.3389/fphar.2023.1279516

Figure Lengend Snippet: Summary of scale-up fermentation data for hbFGF (Mean ± SD, n ≥ 3).

Article Snippet: The polyclonal rabbit anti-human bFGF antibody (Cat.No: 3196S) was acquired from Cell Signaling Technology (United States), while the secondary antibody (goat anti-rabbit lgG/HRP antibody, Cat.No: HS101-01) was procured from Transgene Biotech (Beijing, China).

Techniques:

Journal: Frontiers in Pharmacology

Article Title: Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

doi: 10.3389/fphar.2023.1279516

Figure Lengend Snippet: Process of fermentation of E. coli BL21 (DE3) plysS-mpET3c/hbFGF. (A) Growth curve of hbFGF engineered strain in a 200-L fermentation. (B) SDS-PAGE analysis of the hbFGF expression at the 500-L scale. #1 Lane, non-induced. Lane M, molecular weight marker. (C) Variations in the parameters for hbFGF production at the 500-L scale, including wet cell concentration (OD 600 ), temperature, dissolved oxygen (DO) levels, rotation speed, and air ventilation rate. (D) The relative curves of the specific growth rate, pH, glucose addition, and nitrogen source addition with time in a 500-L fermentation. Black arrows indicate hbFGF.

Article Snippet: The polyclonal rabbit anti-human bFGF antibody (Cat.No: 3196S) was acquired from Cell Signaling Technology (United States), while the secondary antibody (goat anti-rabbit lgG/HRP antibody, Cat.No: HS101-01) was procured from Transgene Biotech (Beijing, China).

Techniques: SDS Page, Expressing, Molecular Weight, Marker, Concentration Assay

Journal: Frontiers in Pharmacology

Article Title: Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

doi: 10.3389/fphar.2023.1279516

Figure Lengend Snippet: Summary of the purification process for hbFGF (Mean ± SD, n = 4).

Article Snippet: The polyclonal rabbit anti-human bFGF antibody (Cat.No: 3196S) was acquired from Cell Signaling Technology (United States), while the secondary antibody (goat anti-rabbit lgG/HRP antibody, Cat.No: HS101-01) was procured from Transgene Biotech (Beijing, China).

Techniques: Purification, Bacteria, Lysis

Journal: Frontiers in Pharmacology

Article Title: Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

doi: 10.3389/fphar.2023.1279516

Figure Lengend Snippet: Identification and analysis of hbFGF in the purification process. (A) SDS-PAGE analysis of hbFGF after ion exchange and affinity chromatography. Lane 1, supernatant. Lane 2, the flow-through sample from CM-Sepharose. Lane 3, 0.36 M NaCl-eluted sample from CM-Sepharose. Lane 4, 2.0 M NaCl-regenerated sample from CM-Sepharose. Lane 5, flow-through fraction from heparin affinity Sepharose. Lane 6, 2.0 M NaCl-eluted sample from heparin affinity Sepharose. Lane 7, 2.0 M NaCl-regenerated sample from SP-Sepharose. Lane 8, purified hbFGF eluted with 0.5 M NaCl from SP-Sepharose. Lane M, molecular weight marker. (B) RP-HPLC and (C) SEC-HPLC analysis of purified hbFGF. (D) The biological activity of hbFGF on NIH-3T3 cells. (E) Analysis of Isoelectric point, (F) Mass spectrum, (G) molecular peptide mapping coverage, and (H) CD spectrum analysis of purified hbFGF. Black arrows indicate hbFGF.

Article Snippet: The polyclonal rabbit anti-human bFGF antibody (Cat.No: 3196S) was acquired from Cell Signaling Technology (United States), while the secondary antibody (goat anti-rabbit lgG/HRP antibody, Cat.No: HS101-01) was procured from Transgene Biotech (Beijing, China).

Techniques: Purification, SDS Page, Affinity Chromatography, Molecular Weight, Marker, Activity Assay

Journal: Frontiers in Pharmacology

Article Title: Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

doi: 10.3389/fphar.2023.1279516

Figure Lengend Snippet: Comparison of hbFGF expressed for various hosts.

Article Snippet: The polyclonal rabbit anti-human bFGF antibody (Cat.No: 3196S) was acquired from Cell Signaling Technology (United States), while the secondary antibody (goat anti-rabbit lgG/HRP antibody, Cat.No: HS101-01) was procured from Transgene Biotech (Beijing, China).

Techniques: Comparison

Journal: Frontiers in Pharmacology

Article Title: Scaling up production of recombinant human basic fibroblast growth factor in an Escherichia coli BL21(DE3) plysS strain and evaluation of its pro-wound healing efficacy

doi: 10.3389/fphar.2023.1279516

Figure Lengend Snippet: The healing effect of hbFGF on a deep second-degree scald wound model in rats. (A) Photos of the skin wound and wound healing rates of STZ-induced SD rats with deep second-degree scald wounds. (B) Results of Masson (scale bar, 200 μm) and IHC staining (scale bar, 50 μm) and the pathological score of a deep second-degree scald wound model in rats. (C) The expression level of FGF-1, TGF-β1, and hydroxyproline in a deep second-degree scald wound model in rats. Compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001, n = 7.

Article Snippet: The polyclonal rabbit anti-human bFGF antibody (Cat.No: 3196S) was acquired from Cell Signaling Technology (United States), while the secondary antibody (goat anti-rabbit lgG/HRP antibody, Cat.No: HS101-01) was procured from Transgene Biotech (Beijing, China).

Techniques: Immunohistochemistry, Expressing, Control

Journal: International Journal of Stem Cells

Article Title: In Vitro Study of Adipose-Derived Mesenchymal Stem Cells Transduced with Lentiviral Vector Carrying the Brain-Derived Neurotrophic Factor Gene

doi: 10.15283/ijsc20038

Figure Lengend Snippet: Western-Blot analyses for murine protein expression of NGF, BDNF, bFGF and CNTF in BDNF-ADSCs and ADSCs. *means statistically significant (p<0.05, n=3), and **means much statistically significant (p<0.01, n=3).

Article Snippet: Primary antibodies used were rabbit anti-p75 NGF receptor polyclonal antibody (bs-7122R; Bioss), rabbit anti-CNTF antibody (bs-1272R; Bioss), rabbit anti-BDNF antibody (ab108319; Abcam), rabbit anti-bFGF antibody (bs-0217R; Bioss).

Techniques: Western Blot, Expressing