<div class="csl-bib-body">
<div class="csl-entry">Kittler, S., Ebner, J., Kopp, J., & Spadiut, O. (2022, March). <i>Small scale mechanical cell disruption: A workflow to screen for ideal disruption conditions for recombinantly produced proteins in E. coli</i> [Poster Presentation]. 7th BioProscale 2022, Berlin, Germany.</div>
</div>
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dc.identifier.uri
http://hdl.handle.net/20.500.12708/153065
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dc.description.abstract
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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dc.description.sponsorship
FFG - Österr. Forschungsförderungs- gesellschaft mbH
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dc.language.iso
en
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dc.subject
cell disruption
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dc.subject
small scale
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dc.subject
workflow
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dc.title
Small scale mechanical cell disruption: A workflow to screen for ideal disruption conditions for recombinantly produced proteins in E. coli
