Coordinator: Sabine Suppmann
Head Recombinant Protein Production
Biochemistry Core Facility
Max-Planck-Institute of Biochemistry
Am Klopferspitz 18| 82151 Martinsried| Germany
phone: +49 8578 2589
Members of the P4EU network use diverse eucaryotic host organisms for recombinant protein production. A survey conducted in order to design a benchmarking study on protein expression in eucaryotic hosts showed major interest in Baculovirus and transient HEK293 expression systems:
The rationale of this benchmarking study was to compare the performance of the protocols used in different protein production facilities for expression of a common set of target proteins. Different target proteins were chosen for expression in insect and HEK293 cells, as the aim of the study was not to compare insect vs HEK293 expression, but to identify the most successful protocols from both expression systems. The comparison of overall protocols, with their many variable parameters made it difficult to pin-point the individual parameter/s crucial for high performance although trends appeared to suggest the importance of certain parameters that can easily be investigated further in follow-up experiments. A positive aspect of this study was that the best performing protocol(s) were identified and can be used as a guide by those colleagues who experience problems, or are setting up a new insect cell platform, to navigate them through the many decisions that have to be made.
The genes chosen for insect cell expression were provided as pFastBac and pFlashBac constructs:
• four intracellular proteins, with previously observed expression levels of < 1mg/L, and with sizes ranging from 68 kDa to 204 kDa. • two secreted proteins, with previously observed expression levels of 0.5 and 10 mg/L, and with sizes ranging from 19kDa to 61 kDa.
The genes chosen for HEK293 expression included
• four secreted proteins, with previously observed expression levels of 0,1 to 10 mg / L and with sizes ranging from 20 kDa to 75 kDa.
• two intracellular proteins, with previously observed expression levels of 0,4 and 1 mg / L, with sizes of 68kDa and 124 kDa.
Participation in the study
• seven labs expressed six insect targets using the pFastBac system.
• seven labs expressed six insect targets using the pFlashBac system.
• seven labs expressed six targets in HEK293 cells.
The study design is described in more detail here: ProjectPage_InsectandHEKbenchmarking_StudyDesign.pdf link
Sample analysis and protein purification was performed at one site to exclude influence of non-expression related parameters on the results.
Results of the study
The first results on expression levels (total lysates) were summarized and presented at the 10th P4EU Meeting in Munich in December 2015. Protein purification and DLS analysis of the purified proteins was perfomed in January 2016 and confirmed the first results with total lysates.
In the HEK data set, productivity in the majority of labs was very similar with two exceptions. Unfortunately, there was no obvious link between productivity of these two groups and the protocolsused, so the HEK exercise proved inconclusive.
In contrast, the insect cell expression results were surprising: 50-fold differences in expression and stability levels for intracellular proteins and 5-fold differences for secreted targets were observed among the different participating groups. Although the successful protocols differed in a variety of parameters they did have in common some reagents and/or procedures that could be responsible for their relatively high performance.
In order to strengthen the interpretation of these results, a second round of experiments including 5 protein production facilities has started in February 2017 aiming at finalizing the entire benchmarking study end of 2017 in a joint publication