Specific expression of LATERAL SUPPRESSOR is controlled by an evolutionarily conserved 3' enhancer.

Aerial plant architecture is largely based on the activity of axillary meristems (AMs), initiated in the axils of leaves. The Arabidopsis gene LATERAL SUPPRESSOR (LAS), which is expressed in well-defined domains at the adaxial boundary of leaf primordia, is a key regulator of AM formation. The precise definition of organ boundaries is an essential step for the formation of new organs in general and for meristem initiation; however, mechanisms leading to these specific patterns are not well understood. To increase understanding of how the highly specific transcript accumulation in organ boundary regions is established, we investigated the LAS promoter. Analysis of deletion constructs revealed that an essential enhancer necessary for complementation is situated about 3.2 kb downstream of the LAS open reading frame. This enhancer is sufficient to confer promoter specificity as upstream sequences in LAS could be replaced by non-specific promoters, such as the 35S minimal promoter. Further promoter swapping experiments using the PISTILLATA or the full 35S promoter demonstrated that the LAS 3' enhancer also has suppressor functions, largely overwriting the activity of different 5' promoters. Phylogenetic analyses suggest that LAS function and regulation are evolutionarily highly conserved. Homologous elements in downstream regulatory sequences were found in all LAS orthologs, including grasses. Transcomplementation experiments demonstrated the functional conservation of non-coding sequences between Solanum lycopersicum (tomato) and Arabidopsis. In summary, our results show that a highly conserved enhancer/suppressor element is the main regulatory module conferring the boundary-specific expression of LAS.

Femtomolar Fab binding affinities to a protein target by alternative CDR residue co-optimization strategies without phage or cell surface display.

In therapeutic or diagnostic antibody discovery, affinity maturation is frequently required to optimize binding properties. In some cases, achieving very high affinity is challenging using the display-based optimization technologies. Here we present an approach that begins with the creation and clonal, quantitative analysis of soluble Fab libraries with complete diversification in adjacent residue pairs encompassing every complementarity-determining region position. This was followed by alternative recombination approaches and high throughput screening to co-optimize large sets of the found improving mutations. We applied this approach to the affinity maturation of the anti-tumor necrosis factor antibody adalimumab and achieved ~500-fold affinity improvement, resulting in femtomolar binding. To our knowledge, this is the first report of the in vitro engineering of a femtomolar affinity antibody against a protein target without display screening. We compare our findings to a previous report that employed extensive mutagenesis and recombination libraries with yeast display screening. The present approach is widely applicable to the most challenging of affinity maturation efforts.