Ira Schieren | PubMed Health

PubMed2006

Type II cadherin ectodomain structures: implications for classical cadherin specificity.

Type I and II classical cadherins help to determine the adhesive specificities of animal cells. Crystal-structure determination of ectodomain regions from three type II cadherins reveals adhesive dimers formed by exchange of N-terminal beta strands between partner extracellular cadherin-1 (EC1) domains. These interfaces have two conserved tryptophan side chains that anchor each swapped strand, compared with one in type I cadherins, and include large hydrophobic regions unique to type II interfaces. The EC1 domains of type I and type II cadherins appear to encode cell adhesive specificity in vitro. Moreover, perturbation of motor neuron segregation with chimeric cadherins depends on EC1 domain identity, suggesting that this region, which includes the structurally defined adhesive interface, encodes type II cadherin functional specificity in vivo.

Amino Acid Sequence↗

PubMed2004

Optimization of protein production in mammalian cells with a coexpressed fluorescent marker.

The expression of mammalian proteins in sufficient abundance and quality for structural studies often presents formidable challenges. Many express poorly in bacterial systems, whereas it can be time consuming and expensive to produce them from cells of higher organisms. Here we describe a procedure for the direct selection of stable mammalian cell lines that express proteins of interest in high yield. Coexpression of a marker protein, such as green fluorescent protein, is linked to that of the desired protein through an internal ribosome entry site in the vector that is transfected into cells in culture. The coexpressed marker is used to select for highly expressing clonal cell lines. Applications are described to a membrane protein, the 5HT2c serotonin receptor, and to a secreted cysteine-rich protein, resistin. Besides providing an expeditious means for producing mammalian proteins for structural work, the resulting cell lines also readily support tests of functional properties and structure-inspired hypotheses.

Animals↗