S48
Abstracts / New Biotechnology 33S (2016) S1–S213
O15-4
Recombinant protein production
Affibody molecules: therapy and in vivo diagnostic applications
O16-1
Torbjörn Gräslund KTH - Royal Institute of Technology, Sweden Affibody molecules are a class of small (6–7 kDa), nonimmunoglobulin based affinity domains that can be generated to specifically interact with desired target proteins. Variants with strong and specific affinity (in the low nanomolar to picomolar range) for a number of targets including IGF-1R, HER2 and the neonatal Fc receptor (FcRn) have been described by us and others, and have been functionalized for use in different medical applications. Radiolabeled affibody molecules have for example been used for PET- and SPECT-based in vivo radionuclide molecular imaging, and have been shown to give exquisite tumor-to-organ/blood ratios in a matter of hours in pre-clinical models of different cancers. Toxin functionalized anti-HER2 receptor affibody molecules have been found to selectively kill cancer cells expressing the receptor and in vivo experiments have shown that careful molecular design can enhance the biodistribution pattern significantly. Furthermore, affibody molecules targeting FcRn have been found to be usable as a novel alternative for half-life extension of attached peptides. The large number of affibody molecules described by us and others with strong and specific affinity for different target proteins, in combination with the variety of functional groups that can be attached, have shown that affibody molecules are a highly robust and flexible class of affinity reagents suitable for a wide range of different medical applications. http://dx.doi.org/10.1016/j.nbt.2016.06.890
Recombinant protein production in Bacillus Jan Maarten van Dijl University of Groningen and University Medical Center, Groningen, The Netherlands Bacillus subtilis and related bacilli are important industrial work horses for protein production due to their ability to secrete commercially relevant proteins, especially technical enzymes, in large amounts into the fermentation broth. However, hyper-production of secretory proteins may lead to induction of the secretion stress-responsive CssR-CssS regulatory system, resulting in elevated expression of quality control proteases, such as HtrA and HtrB. These proteases degrade misfolded proteins secreted via the Sec pathway, resulting in a potential loss of product. An important aim of our ongoing studies is to investigate the secretion stress responses in B. subtilis cells overproducing industrially relevant enzymes in relation to potential expression and secretion bottlenecks. Importantly, recent findings indicate “noisy” expression of htrB in particular cultures, which is indicative of heterogeneous activation of the secretion stress pathway. Our results show that heterogeneous activation of protein secretion stress can be related to distinct sub-populations of high- and low-level enzyme-producing cells in a culture. Importantly, this expression heterogeneity of secretory proteins in B. subtilis can be suppressed by mutagenesis and optimized growth conditions using novel tools that monitor production heterogeneity up to the level of protein secretion per se. http://dx.doi.org/10.1016/j.nbt.2016.06.892
O15-5 A novel platform technology for the generation of fully human bispecific antibodies Simon Krah Merck/TU-Darmstadt, Germany Bispecific antibodies can overcome efficacy issues of conventional monoclonal antibodies and open new strategies for the treatment of several diseases. Nevertheless, the generation of such molecules is difficult because heterodimerization of different heavy chains needs to be realized while light chain misspairing needs to be omitted. Here we describe a unique technology for bispecific antibody generation, wherein in vivo immunization is combined with the power of an in vitro display technology to identify high affinity fully human bispecific antibodies. http://dx.doi.org/10.1016/j.nbt.2016.06.891
O16-2 Expression screening in Escherichia coli using a Golden Gate based modular plasmid library Christine Schreiber ∗ , Tobias Weidner, Denise Salzig, Peter Czermak University of Applied Sciences Mittelhessen, Germany To maximize the protein yield in recombinant protein production, it is crucial to identify the optimal combination of expression plasmid and strain at an early stage of the process development procedure. Currently, most expression screenings are based on a fixed set of pre-assembled expression plasmids, into which the gene of interest is subcloned. We developed an approach to overcome this limitation by the use of a modularly structured plasmid library, within that single plasmid parts – like promoter or affinity tag – can be combined freely to yield a plethora of expression plasmids for the screening. Our system allows for the assembly of expression plasmids based on six variable plasmid parts (promoter, affinity tag, fusion protein, protease cleavage site, target protein and terminator) by Golden Gate cloning. Firstly, the robustness of the cloning system was assessed by assembling 27 expression plasmids. Assembly of all expression plasmids was successful with high cloning efficiencies. Afterwards, an expression screening in 96 well format was carried out to screen for the optimal combination of promoter, fusion protein and expression strain for the expression of three antimicrobial peptides. This led to the identification of high producers with a more than tenfold increase in protein yield compared to the stan-