Besides their function in vertebrate immune systems, antibodies are useful tools in immunohistochemistry, structural biology, diagnostics, and as therapeutics. With a molecular weight of 15 kDa, single-domain antibodies or Nanobodies are the smallest functional antibody units. Nanobodies are derived from heavy chain antibodies, which can be found in camelid species like dromedaries, camels, llamas, or alpacas. Compared to canonical antibodies, Nanobodies provide several advantages like cheap and fast production in bacterial hosts, enhanced stability, low-immunogenicity, and high tissue penetration and are promising molecules for research and as therapeutics. Generation of new Nanobodies is traditionally performed by immunization of camelids with the target antigen. In the past, several Nanobody selection methods have been established that avoid animal immunization entirely. Additionally, these animal-free selection methods, called phage display, yeast display, or mRNA/cDNA display, provide benefits like the ability to generate Nanobodies against nonimmunogenic or toxic targets. Starting point of every animal-free selection is a Nanobody gene library. The design of these Nanobody gene libraries greatly influences the success of a selection. Larger, more variable gene libraries increase the chance of selecting high-affinity binders, while smaller, more conservative library designs usually deliver more well-behaved proteins. For this study, we designed a highly variable Nanobody gene library that uses only few invariant positions, based on multiple sequence alignments of published Nanobody sequences. This Nanobody gene library provides a huge sequence space, which enables selection of Nanobodies for a variety of target antigens. The mRNA/cDNA display selection method is performed entirely ex vivo and allows the use of very large gene libraries. Therefore, this selection method is the right fit for our highly variable Nanobody gene library and was chosen for this project. The proteins retinal guanylate cyclase 1 (retGC1) and its main regulator guanylate cyclase activating protein 1 (GCAP1) are localized in the outer segments of photoreceptor cells and play important roles in the visual phototransduction cycle. At low intracellular Ca2+ concentrations, GCAP1 strongly activates retGC1, which then synthesizes cGMP. This is essential to restore the photoreceptor dark state. Multiple mutations in the retGC1 and GCAP1 genes are associated with rare retinopathies like Leber’s congenital amaurosis or autosomal dominant cone-rod dystrophy. RetGC1- and GCAP1-binding Nanobodies could provide useful tools to gain a better understanding of the function of these two proteins and the mechanisms that influence retinopathies. To date, no retGC1 purification protocol has been published. In this study, we provide a solubilization and purification protocol for retGC1 after expression in a stable TREx293 cell line.
