Structural analysis, multiple kinetic measurements, and binding studies require the production of adequate amounts of soluble target protein. This task is frequently laborious because of low protein-expression levels and high insolubility.
In labs without bioreactors, dozens of shake-flask cultivations need to be run in parallel, and large cell-culture volumes need to be juggled during collection and purification. As a result, many labs desire an alternate cultivation method that can provide enhanced protein yield.
In addition, metabolic engineering and directed evolution of proteins are responsible for the introduction of thousands of strain variants, all of which need to be screened. This process requires high-throughput screening methods, which in turn require a large number of physiologically healthy cells.
Traditional systems for shake-flask or multiwell-plate cultures have been based on cultivation media in batch mode, which has several limitations. For example, Luria-Bertani (LB) and Terrific Broth (TB) media are supplied with the components already in the cultivation. Due to the presence of large quantities of glucose, the bacteria grow rapidly, and the corresponding high respiratory rate exceeds the oxygen-transfer capacity of the cultivation vessel, leading to anaerobic conditions.
In these conditions, the culture media lose control of pH, and dissolved oxygen and the growth-inhibitory effect of overflow metabolites becomes more abundant. These factors contribute to the inability of cultures to reach high cell density and good recombinant protein production.
New Cultivation Method
BioSilta has examined the benefits of fed-batch processes and their ability to produce high cell densities and has developed a novel method to facilitate glucose-limited cell growth in small-scale cultivations. Since the majority of protein production is performed in E. coli, BioSilta developed EnBase® enzyme-based substrate delivery predominantly for bacterial cultivations.
EnBase Flo liquid medium (Figure 1) and EnPresso™ tablets are based on optimized mineral-salt compositions containing a small amount of complex additives and a polysaccharide as the basic carbon source. The introduction of an enzyme, which is provided with the media, degrades the carbon source, thus enabling slow glucose delivery. The rate of glucose release is altered by simply adding more enzyme.
The major advantage of enzyme addition is the ability to control glucose delivery without an external feeding device. EnBase with optimized enzyme dose makes cultivations under glucose-limited conditions possible. The enzymatic control of cell growth by slow substrate-release is ideal for performing fed-batch-like cultivations in shake flasks, tubes, and multiwell plates.