Chickens with a Truncated Light Chain Transgene Express Single-Domain H Chain-Only Antibodies.

H chain-only Igs are naturally produced in camelids and sharks. Because these Abs lack the L chain, the Ag-binding domain is half the size of a traditional Ab, allowing this type of Ig to bind to targets in novel ways. Consequently, the H chain-only single-domain Ab (sdAb) structure has the potential to increase the repertoire and functional range of an active humoral immune system. The majority of vertebrates use the standard heterodimeric (both H and L chains) structure and do not produce sdAb format Igs. To investigate if other animals are able to support sdAb development and function, transgenic chickens (Gallus gallus) were designed to produce H chain-only Abs by omitting the L chain V region and maintaining only the LC region to serve as a chaperone for Ab secretion from the cell. These birds produced 30-50% normal B cell populations within PBMCs and readily expressed chicken sequence sdAbs. Interestingly, the H chains contained a spontaneous CH1 deletion. Although no isotype switching to IgY or IgA occurred, the IgM repertoire was diverse, and immunization with a variety of protein immunogens rapidly produced high and specific serum titers. mAbs of high affinity were efficiently recovered by single B cell screening. In in vitro functional assays, the sdAbs produced by birds immunized against SARS-CoV-2 were also able to strongly neutralize and prevent viral replication. These data suggest that the truncated L chain design successfully supported sdAb development and expression in chickens.

Functional premature polyadenylation signals and aberrant splicing within a recombinant protein coding sequence limit expression.

Recombinant glycoproteins can be produced at high levels in permanently transfected mammalian cells using expression vectors with strong viral promoters. CHO-K1 cell lines developed to produce the recombinant complement activator blocking protein, CAB-2 (a fusion of membrane co-factor protein, MCP, and decay accelerating factor, DAF), showed unexpectedly low expression. Northern blot analysis revealed that in addition to the expected 2300 base CAB-2 mRNA species, these cell lines expressed 790 and 1500 base mRNA species accounting for ~50% and ~10% of the total CAB-2 mRNA, respectively. RT-PCR studies established that the 1500 base species resulted from aberrant splicing from within the DAF region of the CAB-2 coding sequence to a site within the 3' untranslated region. 3' RACE analysis confirmed that the 790 base species resulted from premature polyadenylation at an AATAAA site within the MCP coding region of CAB-2. Another prematurely polyadenylated species, not observed on Northern blots, was observed in the DAF region by 3' RACE. Analysis of human tissues and cell lines revealed that these internal polyadenylation signals in native MCP and DAF coding regions also generated prematurely polyadenylated mRNAs. Genetic modification of these functional RNA processing elements within the CAB-2 gene eliminated the aberrant mRNA species and significantly increased recombinant CAB-2 expression. These results illustrate that protein expression can be limited by aberrant mRNA processing and demonstrate the importance of identifying and eliminating these mRNA processing signals from within coding DNA to maximize recombinant protein expression.

Discovery of diverse and functional antibodies from large human repertoire antibody libraries.

Phage display antibody libraries have a proven track record for the discovery of therapeutic human antibodies, increasing the demand for large and diverse phage antibody libraries for the discovery of new therapeutics. We have constructed naïve antibody phage display libraries in both Fab and scFv formats, with each library having more than 250 billion clones that encompass the human antibody repertoire. These libraries show high fidelity in open reading frame and expression percentages, and their V-gene family distribution, VH-CDR3 length and amino acid usage mirror the natural diversity of human antibodies. Both the Fab and scFv libraries show robust sequence diversity in target-specific binders and differential V-gene usage for each target tested, supporting the use of libraries that utilize multiple display formats and V-gene utilization to maximize antibody-binding diversity. For each of the targets, clones with picomolar affinities were identified from at least one of the libraries and for the two targets assessed for activity, functional antibodies were identified from both libraries.

Kinase partner protein interacts with the LePRK1 and LePRK2 receptor kinases and plays a role in polarized pollen tube growth.

The pollen-specific receptor kinases LePRK1 and LePRK2 have localization and expression profiles that strongly suggest they play roles in pollen germination and tube growth. To identify downstream components of LePRK signaling, we used their cytoplasmic domains (CDs) as baits in yeast two-hybrid screens of a tomato pollen cDNA library. A pollen-specific protein we named kinase partner protein (KPP) interacted with the CDs of both LePRK1 and LePRK2 in yeast and in an in vitro pull-down assay, and with LePRK2 in a co-immunoprecipitation assay. KPP is a peripheral membrane protein and is phosphorylated in pollen. Pollen tubes over-expressing KPP developed balloon-like tips with abnormal cytoplasmic streaming and F-actin arrangements and plants over-expressing KPP exhibited impaired transmission of the transgene through the male. KPP-like genes are found only in plants; the 14 family members in Arabidopsis thaliana exhibit diverse expression patterns and potentially play roles in signaling pathways in other tissues.

LeSTIG1, an extracellular binding partner for the pollen receptor kinases LePRK1 and LePRK2, promotes pollen tube growth in vitro.

As pollen tubes grow through the pistil they are thought to perceive and respond to diverse signals. The tomato pollen-specific receptor kinases LePRK1 and LePRK2 might participate in signaling during pollen tube growth. We previously showed that the extracellular domain of LePRK2 interacts with a pollen protein, LAT52, before but not after pollen germination. To determine whether LePRK2 might have different binding partner(s) after pollen germination, we characterized two more proteins that, like LAT52, were identified in yeast two-hybrid screens using the extracellular domains of LePRK1 and LePRK2 as baits. We show that LeSHY, a leucine-rich repeat protein from pollen, and LeSTIG1, a small cysteine-rich protein from pistil, can bind the extracellular domains of both LePRK1 and LePRK2 in vitro. In vitro binding assays with the extracellular domain of LePRK2 suggested that LeSTIG1 could displace binding of LAT52, consistent with the idea that LePRK1 and LePRK2 might interact with different ligands at different stages of pollen tube growth. Exogenous LeSTIG1 promotes pollen tube growth in vitro. The interaction of these pollen kinases with LeSTIG1 supports the notion that LePRK1 and LePRK2 are involved in mediating pollen-pistil interactions.

New pollen-specific receptor kinases identified in tomato, maize and Arabidopsis: the tomato kinases show overlapping but distinct localization patterns on pollen tubes.

We previously characterized LePRK1 and LePRK2, pollen-specific receptor kinases from tomato (Muschietti et al., 1998). Here we identify a similar receptor kinase from maize, ZmPRK1, that is also specifically expressed late in pollen development, and a third pollen receptor kinase from tomato, LePRK3. LePRK3 is less similar to LePRK1 and LePRK2 than either is to each other. We used immunolocalization to show that all three LePRKs localize to the pollen tube wall, in partially overlapping but distinct patterns. We used RT-PCR and degenerate primers to clone homologues of the tomato kinases from other Solanaceae. We deduced features diagnostic of pollen receptor kinases and used these criteria to identify family members in the Arabidopsis database. RT-PCR confirmed pollen expression for five of these Arabidopsis candidates; two of these are clearly homologues of LePRK3. Our results reveal the existence of a distinct pollen-specific receptor kinase gene family whose members are likely to be involved in perceiving extracellular cues during pollen tube growth.