x ray film santa cruz biotech sc 201696 ifn a2b invivogen rcyc hifna2b luciferase assay system promega e1500  (InvivoGen)

Journal: Microbial Cell Factories

Article Title: Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

doi: 10.1186/s12934-016-0508-5

Figure Lengend Snippet: The design of the IFNα-2b expression construct. IFNα-2b expression is driven by the cbh1 promoter (p) and terminated with the cbh1 terminator (t). IFNα-2b is expressed as a cleavable fusion protein with CBHI. The NVISKR sequence between CBHI and IFNα-2b can be intracellularly cleaved by the KEX2 protease, which resides in the late golgi. The construct contains a hygromycin selection marker (hygR). The construct is targeted to the cbh1 locus via the cbh1 p on the 5′ end and cbh1 3′ flank sequence

Article Snippet: The protein standards were loaded in the gel corresponding to 200, 100 and 50 ng of full length IFNα-2b (Acris Antibodies #AR09043PU-N).

Techniques: Expressing, Construct, Sequencing, Selection, Marker

Journal: Microbial Cell Factories

Article Title: Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

doi: 10.1186/s12934-016-0508-5

Figure Lengend Snippet: Strains created and used in the study

Article Snippet: The protein standards were loaded in the gel corresponding to 200, 100 and 50 ng of full length IFNα-2b (Acris Antibodies #AR09043PU-N).

Techniques:

Journal: Microbial Cell Factories

Article Title: Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

doi: 10.1186/s12934-016-0508-5

Figure Lengend Snippet: Immunoblot detecting IFNα-2b produced by 9-protease deletion strains. Transformants from amp1 (#1–3), slp7 (4), amp2 (#5–7), and sep1 (#9–12) deletion strains were grown in duplicate in 24 well plates in TrMM with 8.6 g/L diammonium citrate, 5.4 g/L NaSO 4 , 100 mM PIPPS, 20 g/L spent grain extract, 40 g/L lactose at pH 4.5, shaking at 28 °C. a The supernatant from day 6 was diluted so that 0.5 µl could be loaded per well. The M577 strain is the parental control (#8). The full length IFNα-2b runs around 17 kDa and the carrier bound material runs at 75 kDa. b Supernatant from day 7 was diluted so that 0.5 µl could be loaded per well. The M577 strain is the parental control (#8)

Article Snippet: The protein standards were loaded in the gel corresponding to 200, 100 and 50 ng of full length IFNα-2b (Acris Antibodies #AR09043PU-N).

Techniques: Western Blot, Produced

Journal: Microbial Cell Factories

Article Title: Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

doi: 10.1186/s12934-016-0508-5

Figure Lengend Snippet: Immunoblot detecting IFNα-2b produced by 9-protease deletion strains. Transformants from pep8 (#2–3) and pep11 (#4–7) deletion strains were grown in duplicate using 24 well plates in TrMM with 8.6 g/L diammonium citrate, 5.4 g/L NaSO 4 , 100 mM PIPPS, 20 g/L spent grain extract, 40 g/L lactose at pH 4.5, shaking at 28 °C. M577 is the parental control (#1). a The supernatant from day 5 was diluted so that 0.5 µl could be loaded per well. The full length IFNα-2b runs around 17 kDa and the carrier bound material runs at 75 kDa. b The supernatant from day 7 was diluted so that 0.5 µl could be loaded per well

Article Snippet: The protein standards were loaded in the gel corresponding to 200, 100 and 50 ng of full length IFNα-2b (Acris Antibodies #AR09043PU-N).

Techniques: Western Blot, Produced

Journal: Microbial Cell Factories

Article Title: Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

doi: 10.1186/s12934-016-0508-5

Figure Lengend Snippet: Immunoblot detecting IFNα-2b production. a M673 (Δ slp7 ) strain treated with protease inhibitors. Cultures were grown in 24 well plates in TrMM plus 20 g/L spent grain extract and 40 g/L lactose, with 8.6 g/L diammonium citrate, 5.4 g/L NaSO 4 , 100 mM PIPPS, pH 4.5 at 28 °C. Duplicate wells were used per treatment. The day 5 and day 7 supernatants were diluted so that 0.2 µl could be loaded per well. 10 µM pepstatin, 20 µM chymostatin, 0.4 mg/ml SBTI, and 20 µM bestatin were used as protease inhibitors. b M674 (Δ amp2 ) strain treated with protease inhibitors. The day 5 and day 7 supernatants were diluted so that 0.2 µl could be loaded per well

Article Snippet: The protein standards were loaded in the gel corresponding to 200, 100 and 50 ng of full length IFNα-2b (Acris Antibodies #AR09043PU-N).

Techniques: Western Blot

Journal: Microbial Cell Factories

Article Title: Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

doi: 10.1186/s12934-016-0508-5

Figure Lengend Snippet: Bioreactor base consumption data (ml) and IFNα-2b expression level data (g/L)

Article Snippet: The protein standards were loaded in the gel corresponding to 200, 100 and 50 ng of full length IFNα-2b (Acris Antibodies #AR09043PU-N).

Techniques: Expressing

Journal: Microbial Cell Factories

Article Title: Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

doi: 10.1186/s12934-016-0508-5

Figure Lengend Snippet: Bioreactor data and IFNα-2b expression level data (g/L)

Article Snippet: The protein standards were loaded in the gel corresponding to 200, 100 and 50 ng of full length IFNα-2b (Acris Antibodies #AR09043PU-N).

Techniques: Expressing

Journal: Microbial Cell Factories

Article Title: Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

doi: 10.1186/s12934-016-0508-5

Figure Lengend Snippet: Immunoblot showing the IFNα-2b production level in the M674 supernatant cultivated in Triab125 and Triab126. Bioreactor cultures were grown in 20 g/L yeast extract, 40 g/L cellulose, 80 g/L cellobiose, and 40 g/L sorbose, 5 g/L (NH 4 ) 2 SO 4 , 5 g/L KH 2 PO 4 at pH 4.5. The Triab126 cultivation was done with SBTI inhibitor feeding (0.4 mg/ml final concentration). The supernatants were diluted so that 0.025 µl could be loaded per well. The protein standards were loaded in the gel corresponding to 200 ng, 100 ng, and 50 ng of full length IFNα-2b. The full length IFNα-2b runs around 17 kDa and the carrier bound material runs at 75 kDa

Article Snippet: The protein standards were loaded in the gel corresponding to 200, 100 and 50 ng of full length IFNα-2b (Acris Antibodies #AR09043PU-N).

Techniques: Western Blot, Concentration Assay

Journal: Microbial Cell Factories

Article Title: Enabling low cost biopharmaceuticals: high level interferon alpha-2b production in Trichoderma reesei

doi: 10.1186/s12934-016-0508-5

Figure Lengend Snippet: Expression systems reported for IFNα-2b production

Article Snippet: The protein standards were loaded in the gel corresponding to 200, 100 and 50 ng of full length IFNα-2b (Acris Antibodies #AR09043PU-N).

Techniques: Expressing, Purification