@article{KrebsRakotoarinoroStechetal.2022, author = {Krebs, Simon K. and Rakotoarinoro, Nathanael and Stech, Marlitt and Zemella, Anne and Kubick, Stefan}, title = {A CHO-based cell-free dual fluorescence reporter system for the straightforward assessment of amber suppression and scFv functionality}, series = {Frontiers in Bioengineering and Biotechnology}, volume = {10}, journal = {Frontiers in Bioengineering and Biotechnology}, publisher = {Frontiers Media}, address = {Lausanne}, issn = {2296-4185}, doi = {10.3389/fbioe.2022.873906}, pages = {15}, year = {2022}, abstract = {Incorporation of noncanonical amino acids (ncAAs) with bioorthogonal reactive groups by amber suppression allows the generation of synthetic proteins with desired novel properties. Such modified molecules are in high demand for basic research and therapeutic applications such as cancer treatment and in vivo imaging. The positioning of the ncAA-responsive codon within the protein's coding sequence is critical in order to maintain protein function, achieve high yields of ncAA-containing protein, and allow effective conjugation. Cell-free ncAA incorporation is of particular interest due to the open nature of cell-free systems and their concurrent ease of manipulation. In this study, we report a straightforward workflow to inquire ncAA positions in regard to incorporation efficiency and protein functionality in a Chinese hamster ovary (CHO) cell-free system. As a model, the well-established orthogonal translation components Escherichia coli tyrosyl-tRNA synthetase (TyrRS) and tRNATyr(CUA) were used to site-specifically incorporate the ncAA p-azido-l-phenylalanine (AzF) in response to UAG codons. A total of seven ncAA sites within an anti-epidermal growth factor receptor (EGFR) single-chain variable fragment (scFv) N-terminally fused to the red fluorescent protein mRFP1 and C-terminally fused to the green fluorescent protein sfGFP were investigated for ncAA incorporation efficiency and impact on antigen binding. The characterized cell-free dual fluorescence reporter system allows screening for ncAA incorporation sites with high incorporation efficiency that maintain protein activity. It is parallelizable, scalable, and easy to operate. We propose that the established CHO-based cell-free dual fluorescence reporter system can be of particular interest for the development of antibody-drug conjugates (ADCs).}, language = {en} } @article{TscheuschnerKaiserLisecetal.2022, author = {Tscheuschner, Georg and Kaiser, Melanie N. and Lisec, Jan and Beslic, Denis and Muth, Thilo and Kr{\"u}ger, Maren and Mages, Hans Werner and Dorner, Brigitte G. and Knospe, Julia and Schenk, J{\"o}rg A. and Sellrie, Frank and Weller, Michael G.}, title = {MALDI-TOF-MS-based identification of monoclonal murine Anti-SARS-CoV-2 antibodies within one hour}, series = {Antibodies}, volume = {11}, journal = {Antibodies}, number = {2}, publisher = {MDPI}, address = {Basel}, issn = {2073-4468}, doi = {10.3390/antib11020027}, pages = {22}, year = {2022}, abstract = {During the SARS-CoV-2 pandemic, many virus-binding monoclonal antibodies have been developed for clinical and diagnostic purposes. This underlines the importance of antibodies as universal bioanalytical reagents. However, little attention is given to the reproducibility crisis that scientific studies are still facing to date. In a recent study, not even half of all research antibodies mentioned in publications could be identified at all. This should spark more efforts in the search for practical solutions for the traceability of antibodies. For this purpose, we used 35 monoclonal antibodies against SARS-CoV-2 to demonstrate how sequence-independent antibody identification can be achieved by simple means applied to the protein. First, we examined the intact and light chain masses of the antibodies relative to the reference material NIST-mAb 8671. Already half of the antibodies could be identified based solely on these two parameters. In addition, we developed two complementary peptide mass fingerprinting methods with MALDI-TOF-MS that can be performed in 60 min and had a combined sequence coverage of over 80\%. One method is based on the partial acidic hydrolysis of the protein by 5 mM of sulfuric acid at 99 degrees C. Furthermore, we established a fast way for a tryptic digest without an alkylation step. We were able to show that the distinction of clones is possible simply by a brief visual comparison of the mass spectra. In this work, two clones originating from the same immunization gave the same fingerprints. Later, a hybridoma sequencing confirmed the sequence identity of these sister clones. In order to automate the spectral comparison for larger libraries of antibodies, we developed the online software ABID 2.0. This open-source software determines the number of matching peptides in the fingerprint spectra. We propose that publications and other documents critically relying on monoclonal antibodies with unknown amino acid sequences should include at least one antibody fingerprint. By fingerprinting an antibody in question, its identity can be confirmed by comparison with a library spectrum at any time and context.}, language = {en} } @article{CzarneckaWeicheltRoedigeretal.2022, author = {Czarnecka, Malgorzata and Weichelt, Ulrike and R{\"o}diger, Stefan and Hanack, Katja}, title = {Novel Anti Double-Stranded Nucleic Acids Full-Length Recombinant Camelid Heavy-Chain Antibody for the Detection of miRNA}, series = {International Journal of Molecular Sciences}, volume = {23}, journal = {International Journal of Molecular Sciences}, edition = {11}, publisher = {MDPI}, address = {Basel, Schweiz}, issn = {1422-0067}, doi = {10.3390/ijms23116275}, pages = {1 -- 18}, year = {2022}, abstract = {The discovery that certain diseases have specific miRNA signatures which correspond to disease progression opens a new biomarker category. The detection of these small non-coding RNAs is performed routinely using body fluids or tissues with real-time PCR, next-generation sequencing, or amplification-based miRNA assays. Antibody-based detection systems allow an easy onset handling compared to PCR or sequencing and can be considered as alternative methods to support miRNA diagnostic in the future. In this study, we describe the generation of a camelid heavy-chain-only antibody specifically recognizing miRNAs to establish an antibody-based detection method. The generation of nucleic acid-specific binders is a challenge. We selected camelid binders via phage display, expressed them as VHH as well as full-length antibodies, and characterized the binding to several miRNAs from a signature specific for dilated cardiomyopathy. The described workflow can be used to create miRNA-specific binders and establish antibody-based detection methods to provide an additional way to analyze disease-specific miRNA signatures.}, language = {en} } @article{ThammSchollReimetal.2017, author = {Thamm, Markus and Scholl, Christina and Reim, Tina and Gruebel, Kornelia and Moeller, Karin and Rossler, Wolfgang and Scheiner, Ricarda}, title = {Neuronal distribution of tyramine and the tyramine receptor AmTAR1 in the honeybee brain}, series = {The journal of comparative neurology}, volume = {525}, journal = {The journal of comparative neurology}, publisher = {Wiley}, address = {Hoboken}, issn = {0021-9967}, doi = {10.1002/cne.24228}, pages = {2615 -- 2631}, year = {2017}, abstract = {Tyramine is an important neurotransmitter, neuromodulator, and neurohormone in insects. In honeybees, it is assumed to have functions in modulating sensory responsiveness and controlling motor behavior. Tyramine can bind to two characterized receptors in honeybees, both of which are coupled to intracellular cAMP pathways. How tyramine acts on neuronal, cellular and circuit levels is unclear. We investigated the spatial brain expression of the tyramine receptor AmTAR1 using a specific antibody. This antibody detects a membrane protein of the expected molecular weight in western blot analysis. In honeybee brains, it labels different structures which process sensory information. Labeling along the antennal nerve, in projections of the dorsal lobe and in the gnathal ganglion suggest that tyramine receptors are involved in modulating gustatory and tactile perception. Furthermore, the ellipsoid body of the central complex and giant synapses in the lateral complex show AmTAR1-like immunoreactivity (AmTAR1-IR), suggesting a role of this receptor in modulating sky-compass information and/or higher sensor-motor control. Additionally, intense signals derive from the mushroom bodies, higher-order integration centers for olfactory, visual, gustatory and tactile information. To investigate whether AmTAR1-expressing brain structures are in vicinity to tyramine releasing sites, a specific tyramine antibody was applied. Tyramine-like labeling was observed in AmTAR1-IR positive structures, although it was sometimes weak and we did not always find a direct match of ligand and receptor. Moreover, tyramine-like immunoreactivity was also found in brain regions without AmTAR1-IR (optic lobes, antennal lobes), indicating that other tyramine-specific receptors may be expressed there.}, language = {en} }