Journal: Bioscience, biotechnology, and biochemistry

Article Title: Production of brain-derived neurotrophic factor in Escherichia coli by coexpression of Dsb proteins.

doi: 10.1271/bbb.66.344

Figure Lengend Snippet: Fig. 1. Detection of BDNF in Soluble and Insoluble Forms by Immunoblotting. Supernatant samples obtained in 350 ml of culture broth and precipitate samples obtained in 70 ml of culture broth were elec- trophoresed at 20 mA for 2 h using a 0.1z SDS-15z PAGE gel, after boiling for 5 min with a 10 mM Tris-HCl buŠer (pH 6.8) contain- ing 20z glycerol, 1z SDS, 1z 2-mercaptoethanol, and 0.02z bromophenol blue. Glycerol medium was used. IA (3-b-indoleacrylic acid) and casamino acids were added to induce BDNF expression at 2-h cultivation. Panel A: Supernatant (soluble BDNF). Panel B: Precipitate (insoluble BDNF). M, marker; lane 1, E. coli HB101 harboring pTRSBDNF; lane 2, E. coli HB101 harboring pTRSBDNF with addition of L-arabinose; lane 3, E. coli HB101 harboring pTRSBDNF and pDbAB1 with addition of L-arabinose; lane 4, E. coli HB101 harboring pTRSBDNF and pDbAC1 with addition of L-arabinose; lane 5, E. coli HB101 harboring pTRSBDNF and pDbBD1 with addition of L-arabinose; lane 6, E. coli HB101 harboring pTRSBDNF and pDbCD1 with addition of L-arabinose; lane 7, E. coli HB101 harboring pTRSBDNF and pDbABCD1 with addition of L-arabinose; lane 8, 0.05-mg authentic BDNF; lane 9, 0.1-mg authentic BDNF.

Article Snippet: BDNF was detected by using anti-rab- bit BDNF polyclonal antibodies (Santa Cruz Biotechnology, USA) and a goat anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad, USA).3) The BDNF was measured using an imaging densitometer (Model GS-710, Bio-Rad, USA).

Techniques: Western Blot, Expressing, Marker

Journal: Bioscience, biotechnology, and biochemistry

Article Title: Production of brain-derived neurotrophic factor in Escherichia coli by coexpression of Dsb proteins.

doi: 10.1271/bbb.66.344

Figure Lengend Snippet: Fig. 3. Expression Level of DsbC Protein. Panel A: Glucose medium. The results of BDNF production are indicated in Fig. 2A. Panel B: Glycerol medium. The results of BDNF production are indicated in Fig. 4A. Dsb proteins were induced by addition of 0.2 gWl L-arabinose at the beginning of cultivation. IA and casamino acids were not added to the culture. BSA was used as a protein standard.

Article Snippet: BDNF was detected by using anti-rab- bit BDNF polyclonal antibodies (Santa Cruz Biotechnology, USA) and a goat anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad, USA).3) The BDNF was measured using an imaging densitometer (Model GS-710, Bio-Rad, USA).

Techniques: Expressing

Journal: Bioscience, biotechnology, and biochemistry

Article Title: Production of brain-derived neurotrophic factor in Escherichia coli by coexpression of Dsb proteins.

doi: 10.1271/bbb.66.344

Figure Lengend Snippet: Fig. 2. BDNF Production in Soluble and Insoluble Forms with Dsb Coexpression in Glucose Medium. Glucose medium was used. Dsb proteins were induced by addition of 0.2 gWl L-arabinose at the beginning of cultivation. Panel A: IA and casamino acids were not added to the culture. Panel B: IA and casamino acids were added to the culture at 2 h of cultivation. , soluble BDNF; , insoluble BDNF. pTRSBDNF, E. coli HB101 harboring pTRSBDNF; pTRSBDNF+Ara, E. coli HB101 harboring pTRSBDNF with addition of L-arabinose; pDbAB1, E. coli HB101 harboring pTRSBDNF and pDbAB1 with addition of L-arabinose; pDbAC1, E. coli HB101 harboring pTRSBDNF and pDbAC1 with addition of L-arabinose; pDbBD1, E. coli HB101 harboring pTRSBDNF and pDbBD1 with addition of L-arabinose; pDbCD1, E. coli HB101 harboring pTRSBDNF and pDbCD1 with addition of

Article Snippet: BDNF was detected by using anti-rab- bit BDNF polyclonal antibodies (Santa Cruz Biotechnology, USA) and a goat anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad, USA).3) The BDNF was measured using an imaging densitometer (Model GS-710, Bio-Rad, USA).

Techniques:

Journal: Bioscience, biotechnology, and biochemistry

Article Title: Production of brain-derived neurotrophic factor in Escherichia coli by coexpression of Dsb proteins.

doi: 10.1271/bbb.66.344

Figure Lengend Snippet: Fig. 4. BDNF Production in Soluble and Insoluble Forms with Dsb Coexpression in Glycerol Medium. Glycerol medium was used. Dsb proteins were induced by addition of 0.2 gWl L-arabinose at the beginning of cultivation. Panel A: IA and casamino acids were not added to the culture. Panel B: IA and casamino acids were added to the culture at 2 h of cultivation. , soluble BDNF; , insoluble BDNF.

Article Snippet: BDNF was detected by using anti-rab- bit BDNF polyclonal antibodies (Santa Cruz Biotechnology, USA) and a goat anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad, USA).3) The BDNF was measured using an imaging densitometer (Model GS-710, Bio-Rad, USA).

Techniques:

Journal: Bioscience, biotechnology, and biochemistry

Article Title: Production of brain-derived neurotrophic factor in Escherichia coli by coexpression of Dsb proteins.

doi: 10.1271/bbb.66.344

Figure Lengend Snippet: Fig. 5. Soluble BDNF Production Based on Biological Activity of BDNF. Soluble BDNF production was estimated from the biological activity for neurite outgrowth of embryonic chick DRG neurons. Soluble BDNF fractions (Fig. 4) obtained from E. coli cells in glycerol medium were used. Panel A: IA and casamino acids were not added to the culture. Panel B: IA and casamino acids were added to the culture at 2 h of cultivation.

Article Snippet: BDNF was detected by using anti-rab- bit BDNF polyclonal antibodies (Santa Cruz Biotechnology, USA) and a goat anti-rabbit IgG horseradish peroxidase conjugate (Bio-Rad, USA).3) The BDNF was measured using an imaging densitometer (Model GS-710, Bio-Rad, USA).

Techniques: Activity Assay