Strain list.

<p>Strain list.</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

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

Antibody stability in culture supernatant.

<p>Immunoblot showing improved stability of antibody heavy (HC) and light chain (LC). Three model antibodies were tested in large shake flask supernatant (M181 Δ<i>pep1</i> and M124) and M44 fermentation supernatant. The supernatants were each diluted so that the total protein was 2 mg/ml for each culture and incubated with antibody for 18 hours at 37°C and pH 5.5.</p

Protease deletion strains grow faster than parental M124 strain.

<p>Parallel fermentation data (DASGIP Bioreactor System) showing the growth curves for the M124 strain (wt) and the M277 (Δ3), M307 (Δ4), M369 (Δ5), M396 (Δ6), and M486 (Δ7) multiple deletion strains in complex media. Growth was monitored over 3 days following the Aber Futura biomass signal (capacitance) and CO₂ transfer rates. (Note: M277 CO₂ transfer rates are not displayed due to defective off gas monitoring for this single reactor.)</p

Consecutive deletion of proteases resulted in dramatic reduction in protease activity.

<p>Normalized protease activity data from culture supernatants from each of the protease deletion supernatants and the parent strain M124. Protease activity is against a casein substrate. The 6 protease deletion strain has only 6% of the wild type parent strain and the 7 protease deletion strain protease activity went down to 4% based upon casein substrate activity. Wild-type (M124), Δ1 (M181), Δ2 (M219), Δ3 (M277), Δ4 (M307), Δ5 (M369), Δ6 (M396), and Δ7 (M486).</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

Aspartic protease purification and activity.

<p>Fig 1A. Coomassie stained PAGE gel showing fractions eluted from pepstatin affinity purification from the M44 strain. The purified aspartic proteases run around 42 kDa. Fraction numbers are listed above the lanes. Fig 1B. Commassie stained PAGE gel showing aspartic protease degradation of human IgG with and without pepstatin A inhibitor (100 μM). Purified fraction #3 (F3) from the M44 strain was incubated with IgG in sodium citrate buffer at pH 5.5 for 20 hours at 37°C. Nonreduced gel showing the whole assembled antibody and degradation products. The fully assembled antibody runs above 150 kDa. Fig 1C. Commassie stained PAGE gel visualizing the peak fractions from aspartic protease purifications done on the M169 parental strain and M182 Δ<i>pep1</i> strain. The control aspartic protease sample was from a previous purification. The predominant band at 42 kDa corresponds to <i>pep1</i>.</p