Nanobodies are single domain antibodies derived from the variable regions of Camelidae atypical immunoglobulins. They show great promise as high affinity reagents for research, diagnostics and therapeutics due to their high specificity, small size (~15 kDa) and straightforward bacterial expression. However, identification of repertoires with sufficiently high affinity has proven time consuming and difficult, hampering nanobody implementation. Here, we present a rapid, straightforward approach that generates large repertoires of readily expressible recombinant nanobodies with high affinities and specificities against a given antigen. We demonstrate the efficacy of this approach through the production of large repertoires of nanobodies against two antigens, GFP and mCherry, with Kd values into the sub-nanomolar range. After mapping diverse epitopes on GFP, we were also able to design ultra-high affinity dimeric nanobodies with Kds down to ~30 pM. The approach presented is well-suited for the routine production of high affinity capture reagents for various biomedical applications.

Inositol pyrophosphates are a diverse group of high-energy signaling molecules whose cellular roles remain an active area of study. We report a previously uncharacterized class of inositol pyrophosphate synthase and find it is identical to yeast Vip1 and Asp1 proteins, regulators of actin-related protein-2/3 (ARP 2/3) complexes. Vip1 and Asp1 acted as enzymes that encode inositol hexakisphosphate (IP6) and inositol heptakisphosphate (IP7) kinase activities. Alterations in kinase activity led to defects in cell growth, morphology, and interactions with ARP complex members. The functionality of Asp1 and Vip1 may provide cells with increased signaling capacity through metabolism of IP6.

). Northern blot analysis indicates that human VIP1 is expressed in a variety of tissues and is enriched in skeletal muscle, heart, and brain. The subcellular distribution of tagged human VIP1 is indicative of a cytoplasmic non-membrane localization pattern. We also characterized human and mouse VIP2, an additional gene product with nearly 90% similarity to VIP1 in the kinase domain, and observed both IP 6 and IP 7 kinase activities. Our data demonstrate that human VIP1 and VIP2 function as IP 6 and IP 7 kinases that act along with the IP6K/Kcs1-class of kinases to convert IP 6 to IP 8 in mammalian cells, a process that has been found to occur in response to various stimuli and signaling events. Inositol phosphates (IPs)2 are a diverse group of regulatory molecules involved in a variety of intracellular signaling pathways. Stimulation of cells results in the production of IPs through the cleavage of phosphoinositides by phosphoinositide-specific phospholipase C (1-3). Many additional IPs, such as inositol tetrakisphosphate (IP 4 ), inositol pentakisphosphate (IP 5 ), and inositol hexakisphosphate (IP 6 ), are generated through the subsequent action of several classes of evolutionarily conserved inositol phosphate kinases (IPKs) (2, 4 -7). Genetic and biochemical studies of the IPKs have linked their IP products to processes including metal chelation, ion channel regulation, transcription, chromatin remodeling, nuclear mRNA export, apoptosis, RNA editing, phosphate sensing, auxin signaling, and proper organism development (reviewed in Refs. 1-4, 6, 8).Soluble IP molecules also serve as precursors to several species of diphosphoinositol phosphates (PP-IPs), commonly referred to as inositol pyrophosphates. PP-IPs were first reported and characterized in Dictyostelium discoideum and mammalian cells and are distinguished by the presence of one or more pyrophosphate groups on the inositol ring (9 -11). Two classes of evolutionarily conserved kinases have been identified as required for the production of PP-IP molecules.An IP 6 kinase (IP6K) class of enzymes, Kcs1 in Saccharomyces cerevisiae and IHPK1, IHPK2, and IHPK3 in mammals, was found to convert IP 6 to diphosphoinositol pentakisphosphate, also known as PP-IP 5 or IP 7 (12, 13), and to phosphorylate IP 5 to produce PP-IP 4 . Loss of Kcs1 in budding yeast results in defects in the response to osmotic stress, regulation of telomere length, vacuolar biogenesis, endocytosis, and other cellular processes (13-18). PP-IP 5 has also been shown in vitro to act as a phosphate donor, capable of phosphorylating proteins directly, in a non-enzymatic process (19); however, evidence that this occurs in cells awaits further study (20). Recent work has also indicated that IP6K activity participates in the synthesis of bisdiphosphoinositol tetrakisphosphate (PP 2 -IP 4 or IP 8 ), a more highly phosphorylated PP-IP species containing two pyrophosphate groups. Studies have demonstrated that PP 2 -IP 4 levels change in response to osmotic and heat stress in both yeast and mammalian c...