16.食道静脈瘤出血に対する経皮経肝塞栓術の経験(第638回千葉医学会例会・第1内科教室同門会例会)

Additional file 1: Table S1–S21. Lists of primers used in PCR experiments

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Additional file 3: Figure S2. The kex2 expression cassette targeted to the pep3 locus removed from plasmid pTTv205 by PmeI digestion

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Additional file 4: Figure S3. Protease activity measurements of purified fractions from culture supernatant sampled on day 4 and 5. The fractions from the pepstatin columns were measured at pH 4.5 and the SBTI fractions at pH 5.5. The protease activity on day 5 was higher than on day 4

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Additional file 1: Table S1–S21. Lists of primers used in PCR experiments

Strain list.

<p>Strain list.</p

MAB02 zymogram activity of protease deletion strain supernatants.

<p>White regions on the blue stained gel indicate an area of protease activity against MAB02. The major activity occurs between 65–90 kDa and a smaller protease activity occurs at 28 kDa. Culture supernatant samples from day 7 were diluted 1:2 in sodium citrate buffer (50 mM, pH 5.5) and 30 μl was loaded into a 12% zymogram SDS PAGE gel containing MAB02. The M124 parental strain, M181 (Δ<i>pep1</i>), M219 (Δ<i>pep1</i>Δ<i>tsp1</i>), and M277 (Δ<i>pep1</i>Δ<i>tsp1</i>Δ<i>slp1</i>) supernatants were assayed.</p

Stability of model therapeutic proteins.

<p>Undiluted supernatant from the M396 (Δ6) strain was used at pH 4.2 for spiking in pure model proteins (0.05 μg/μl). 50 mM sodium citrate pH 4.0 spiked with model proteins (0.05 μg/μl) is shown as a buffer control. The spiked supernatant and control were incubated for 20 hours at 37°C. 10 μl of each sample was loaded into 12 or 18% SDS PAGE gels. The IFNα2b ran at 19.4 kDa, the IGF1 ran at 7.5 kDa, and the MAB01 heavy chain ran at 50 kDa.</p

Total protease activity of protease deletion culture supernatants.

<p>The relative ability of each culture supernatant to degrade succinylated casein is shown. The supernatants were diluted to 2 mg/ml total protein in 50 mM sodium citrate, pH 5.5 before being assayed. 50 μl of diluted supernatant was loaded into a 96 well plate and 50 μl of succinylated casein was added to begin the reaction. The M181 (Δ1), M219 (Δ2), M277 (Δ3) strain supernatants display less protease activity compared to the M124 control strain.</p

Inhibitor treatments stabilized antibody heavy chain.

<p>Immunoblot showing the rituximab heavy chain fragments created by M44 supernatant proteases. The fed batch supernatant was diluted to 6 mg/ml in sodium citrate buffer pH 5.5 and incubated with 0.05 μg/μl of rituximab, 100 μM chymostatin, 1 mg/ml SBTI, or a combination of both inhibitors at 37°C for up to 19 hours.</p

Aminobenzamidine purified serine proteases degraded antibody.

<p>Fig 4A. Proteases from the aminobenzamidine affinity purification fraction #4 degraded human IgG. Samples were taken at 0 min and 20 hours after incubating at pH 5.5 and 37°C. The IgG control was antibody in buffer incubated for 20 hours. Immunoblotting was done to detect the heavy and light chain of the nonreduced IgG. The full length antibody runs around 150 kDa. Fig 4B. Zymogram analysis of MAB02 antibody protease degradation from aminobenzamidine column purified fractions (F2-F4) and from M44 supernatant samples. Purified samples were incubated with and without PMSF serine protease inhibitor at pH 5.5. White band indicate degradation of MAB02 antibody. Protease activity came from 65 and 29 kDa, as the arrows indicate.</p