Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light‐driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochrome b6f complex towards photosystem I (PSI). This membrane protein complex is responsible for the light‐driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP+ reductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI−Fd and PSI−Fd−FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C‐terminus of the PSI−PsaE subunit in the cyanobacterium Synechocystis sp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed in Escherichia coli. Isolated Im7−PSI was coupled with the E7−Fd or E7−Fd−FNR fusion proteins through high‐affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.

Photosynthetic organisms like plants, algae, and cyanobacteria use light for the regeneration of dihydronicotinamide dinucleotide phosphate (NADPH). The process starts with the light-driven oxidation of water by photosystem II (PSII) and the released electrons are transferred via the cytochromeb6fcomplex towards photosystem I (PSI). This membrane protein complex is responsible for the light-driven reduction of the soluble electron mediator ferredoxin (Fd), which passes the electrons to ferredoxin NADP+oxidoreductase (FNR). Finally, NADPH is regenerated by FNR at the end of the electron transfer chain. In this study, we established a clickable fusion system for in vitro NADPH regeneration with PSI-Fd and PSI-Fd-FNR, respectively. For this, we fused immunity protein 7 (Im7) to the C-terminus of the PSI-PsaE subunit in the cyanobacteriumSynechocystissp. PCC 6803. Furthermore, colicin DNase E7 (E7) fusion chimeras of Fd and FNR with varying linker domains were expressed inE. coli. Isolated Im7-PSI was coupled with the E7-Fd or E7-Fd-FNR fusion proteins through high-affinity binding of the E7/Im7 protein pair. The corresponding complexes were tested for NADPH regeneration capacity in comparison to the free protein systems demonstrating the general applicability of the strategy.

Background It is believed, that one of the first events in the progression of Alzheimer’s Disease is the misfolding of the Amyloid Beta (Aβ) Peptide. This event takes place up to 20 years before the clinical onset of the disease. Therefore, biosensors detecting the misfolding of Aβ are especially useful for the early and specific detection of Alzheimer’s Disease. In recent studies, our biosensor based on ATR‐FTIR spectroscopy performed with an overall accuracy of 86% [1,2,3]. This system is robust and reliable but the user input required is very high because each sensor can only be used once. To overcome these issues and increase the automation capacity, a regenerative system is urgently needed. Method To achieve higher throughput of this system, we introduced immunoglobulin binding proteins (Protein A and Protein G) as capture for antibody immobilization. Protein A and Protein G are able to recognize and bind the constant region of different IgG antibodies in a non‐covlant manner. This allows multiple measurements within the same system by performing multiple binding and elution cycles. Result Protein A and Protein G can be attached covalently to the sensor without losing its ability to bind IgG antibodies [4]. The antibody binding and elution is monitored by ATR‐FTIR spectroscopy and validated using fluorescence spectroscopy. It is possible to perform at least 35 cycles of antibody binding and elution over 7 days without loss in diagnostic accuracy. The sensor is still able to extract different biomarkers like Aβ and the Tau Protein from CSF with highly comparable results compared to the recently used system. Conclusion The development of a regenerative immuno‐infrared‐sensor is crucial to reduce the user input and to increase the throughput of this method. Compared to assays where the capture antibody is covalently immobilized, the regenerative system has a factor 6 increased thoughput. References: (1) Nabers, A. et al. (2016) J. Biophotonics, 9. 224‐234. (2) Nabers, A. et al. (2018) EMBO Mol. Med. 10, e8763. (3) Nabers, A et al. (2019) Alzheimer’s Dement. Diagnosis, Assess. Dis. Monit., 11, 257‐263. (4) Budde, B. et al. (2019) ACS Sens. 4, 1851‐1856.