We report the improved production of recombinant proteins in E. coli , reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales.Expression levels using this approach have reached as high as 55% of total cellular protein.Initial use of the method in instrumented fed batch fermentations enables cell densities of~30 grams dry cell weight (gCDW) per liter and protein titers up to 8.1+/-0.7 g/L (~270 mg/gCDW).The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 L (384 well plates), 100 L (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach~5-7 gCDW per liter, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation.
Griffithsin, a broad-spectrum antiviral lectin, has potential to prevent and treat numerous viruses including HIV, HCV, HSV, SARS-CoV, and SARS-CoV-2. For these indications, the annual demand for Griffithsin could reach billions of doses and affordability is paramount. We report the lab-scale validation of a bioprocess that supports production volumes of >20 tons per year at a cost of goods sold below $3,500/kg. Recombinant expression in engineered E. coli enables Griffithsin titers ∼2.5 g/L. A single rapid precipitation step provides > 90% yield with 2-, 3-, and 4-log reductions in host cell proteins, endotoxin, and nucleic acids, respectively. Two polishing chromatography steps remove residual contaminants leading to pure, active Griffithsin. Compared to a conventional one this process shows lower costs and improved economies of scale. These results support the potential of biologics in very large-scale, cost-sensitive applications such as antivirals, and highlight the importance of bioprocess innovations in enabling these applications.
We report the improved production of recombinant proteins in E. coli , reliant on tightly controlled autoinduction, triggered by phosphate depletion in stationary phase. The method, reliant on engineered strains and plasmids, enables improved protein expression across scales.Expression levels using this approach have reached as high as 55% of total cellular protein.Initial use of the method in instrumented fed batch fermentations enables cell densities of 10 grams dry cell weight (gCDW) per liter and protein titers up to 2.7+/-0.2 g/L (270 mg/gCDW).The process has also been adapted to an optimized autoinduction media, enabling routine batch production at culture volumes of 20 L (384 well plates), 100 L (96 well plates), 20 mL and 100 mL. In batch cultures, cells densities routinely reach~5-7 gCDW per liter, offering protein titers above 2 g/L. The methodology has been validated with a set of diverse heterologous proteins and is of general use for the facile optimization of routine protein expression from high throughput screens to fed-batch fermentation Highlights :• Stationary phase protein expression results in high titers.• Autoinduction by phosphate depletion enables protein titers from 2-3 g/L.• Autoinduction has been validated from 384 well plates to instrumented bioreactors.
Objective External distraction appears to affect at least 6–9% of distraction-affected motor vehicle collisions. Billboards may be good models for studying external distraction in general, and it is also desirable to understand billboard-related distraction per se. However, there has not yet been a clear consensus on the scope of billboard-related distraction or its dynamics with respect to characteristics of drivers, billboards, traffic, and the roadway. To narrow these knowledge gaps, a systematic literature review was conducted on billboard-related changes in driver visual behavior. Methods A systematic literature search yielded 443 results, of which 8 studies met all inclusion criteria. Five studies meeting all inclusion criteria were later identified and added. Results were analyzed in terms of 4 categories of visual behavior: (1) gaze variability (GV), glance pattern activity (GPA), and percentage of time spent glancing at the forward roadway; (2) glances at unexpected drive-relevant stimuli; (3) glances at expected drive-relevant stimuli; and (4) glances at billboards. Results There was considerable evidence that about 10–20% of all glances at billboards were ≥0.75 s, that active billboards drew more glances and more long glances (≥0.75 s, ≥2.0 s) than passive billboards but did not attract a longer average glance, and that there was large variability among individual billboards within categories (e.g., active vs. passive). The extent to which billboards attracted glances ≥ 2.0 s was uncertain. There was tentative evidence that billboards did not affect GPA, glances at expected drive-relevant stimuli, or the proportion of time drivers spent glancing at the forward roadway and that they did affect vertical GV and glances at unexpected drive-relevant stimuli. Conclusions Generally, billboard-related distraction appeared to be minor and regulated by drivers as the demands of the driving task changed. However, this review’s findings suggest that this may not be true in all cases. Future research should emphasize the tails of the distribution in addition to average cases, in terms of both the analysis of visual behavior and the complexity of driving tasks. Further research is also needed to understand the effects of billboard design, driver characteristics, and road and traffic context.
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