Kittler, S., Ebner, J., Kopp, J., & Spadiut, O. (2022, March). Small scale mechanical cell disruption: A workflow to screen for ideal disruption conditions for recombinantly produced proteins in E. coli [Poster Presentation]. 7th BioProscale 2022, Berlin, Germany.
The majority of processes using Escherichia coli require cell disruption to release the intracellularly produced target protein. Several mechanical and non-mechanical methods are commonly applied. The most frequently employed method in industry, high pressure homogenization, requires large sample volumes (>20 mL), which is problematic for small scale screening. Hence, for screening experiments enzymatic methods (e.g. Lysozyme) are often used. These methods lack in reproducibility, scalability and might influence purity patterns of samples due to the additional enzyme addition. Thus, the need for a small scale mechanical disruption method is given. Even tough an ultrasonic homogenization device is already reported in literature, application of standardized protocols might lead to fluctuating recoveries for various proteins. Depending on the expression state (inclusion body/soluble), target protein characteristics and localization (cytoplasm/periplasm) different conditions for ultrasonic cell disruption are required. To test this hypothesis, we investigated the factors power input, sonication time and cycles of sonication for three different target proteins. Disruption efficiency was determined in comparison to high pressure homogenization and enzymatic cell lysis by applying a variety of analytical methods. Based on this study, we show that (i) the ultrasonic lance is suited for mechanical cell disruption in small scale screening and (ii) a workflow has been developed to screen for suitable cell disruption conditions, measuring DNA content (absorbance at 260 nm) and protein concentration using an established RPHPLC method.
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Project title:
EIS für Fabs: 874206 (FFG - Österr. Forschungsförderungs- gesellschaft mbH)
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Research Areas:
Efficient Utilisation of Material Resources: 43% Beyond TUW-research foci: 37% Modeling and Simulation: 20%