Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Recombinant Human Brain-Derived Neurotrophic Factor in Escherichia coli Through the Engineering of Its Pro-Region

doi: 10.3390/ijms252413425

Figure Lengend Snippet: The rhProBDNF amino acid sequence as codified in the expression vector. The pro-region is reported in black, whereas the mature BDNF is reported in green. In red, the wild-type hinge region corresponding to the furin cleavage site in between the two domains. The numeration starts at the first methionine substituting the natural protein leader sequence.

Article Snippet: Membranes were then incubated with a primary human anti-BDNF polyclonal antibody (Proteintech, Rosemont, IL, USA, code # 25699-1-AP) diluted (1:1000) in 5% non-fat dry milk in PBS-T 0.1% overnight at 4 °C.

Techniques: Sequencing, Expressing, Plasmid Preparation

Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Recombinant Human Brain-Derived Neurotrophic Factor in Escherichia coli Through the Engineering of Its Pro-Region

doi: 10.3390/ijms252413425

Figure Lengend Snippet: An SP Sepharose typical chromatogram of the wild-type ProBDNF digested by trypsin. The column has been eluted isocratically with a salt step. Fraction numbers are reported at the bottom of the graph. The mature BDNF protein was expected in fractions 6 and 7. Note that fraction 10, corresponding to column washing with 2 M NaCl, has a maximum absorbance of 2000 mAU. The blue line identifies the UV detection at 280 nm, the orange line refers to the measured conductivity.

Article Snippet: Membranes were then incubated with a primary human anti-BDNF polyclonal antibody (Proteintech, Rosemont, IL, USA, code # 25699-1-AP) diluted (1:1000) in 5% non-fat dry milk in PBS-T 0.1% overnight at 4 °C.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Recombinant Human Brain-Derived Neurotrophic Factor in Escherichia coli Through the Engineering of Its Pro-Region

doi: 10.3390/ijms252413425

Figure Lengend Snippet: Representative rhBDNF analysis results after SP elution step. The presence of rhBDNF and its hyperdigested form (ΔHSDPAR) have been measured in different fractions by RP-UPLC analysis. In fractions 6 and 7, the correctly processed protein is heavily contaminated by its hyperdigested form. Fraction 10, containing most of the initial material, mainly corresponds to enzymatic digestion variant (EDG) forms VRR-BDNF and R-BDNF, as determined by mass analysis.

Article Snippet: Membranes were then incubated with a primary human anti-BDNF polyclonal antibody (Proteintech, Rosemont, IL, USA, code # 25699-1-AP) diluted (1:1000) in 5% non-fat dry milk in PBS-T 0.1% overnight at 4 °C.

Techniques: Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: Efficient Production of Recombinant Human Brain-Derived Neurotrophic Factor in Escherichia coli Through the Engineering of Its Pro-Region

doi: 10.3390/ijms252413425

Figure Lengend Snippet: The rhProBDNF amino acid sequence and trypsin cleavage sites. Lysine (K) residues are highlighted in green, while arginines (R) in yellow. Red-highlighted residues are P 1 ’ amino acids impeding trypsin cleavage. Black sequence refers to the Pro-peptide region, while green sequence to mature BDNF.

Article Snippet: Membranes were then incubated with a primary human anti-BDNF polyclonal antibody (Proteintech, Rosemont, IL, USA, code # 25699-1-AP) diluted (1:1000) in 5% non-fat dry milk in PBS-T 0.1% overnight at 4 °C.

Techniques: Sequencing