Morino K et al. (2001) J Immunol Methods "Antibody fusions with fluorescent proteins: a versatile reagent for profiling protein expression."

Shinohara M, Kohara Y, Kurosawa Y, Katsumi H, Ukai Y, Iba Y, Morino K, Akahori Y

[J Immunol Methods, 2001]

We developed a system by which antibodies, fused to fluorescent proteins with different wavelengths, can be prepared within a month against various antigens. An antibody library composed of a large number of single-chain Fv-CL fragment was constructed by means of a phage-display system. The constructs were designed to facilitate changing of the protein forms by simple enzyme manipulation. In the present study, we adopted a molecular form of antibody in which a single-chain Fv-CL fragment is fused with a green fluorescent protein (GFP) or red fluorescent protein (RFP). In addition, a His-tag was inserted between CL and GFP (or RFP). We describe the utility of this system using Caenorhabditis elegans embryo as a model.

Dorfmueller HC et al. (2012) Acta Crystallogr D Biol Crystallogr "Structural and biochemical characterization of a trapped coenzyme A adduct of Caenorhabditis elegans glucosamine-6-phosphate N-acetyltransferase 1."

Rao FV, Attrill H, Dorfmueller HC, van Aalten DM, Fang W, Blair DE

[Acta Crystallogr D Biol Crystallogr, 2012]

Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) produces GlcNAc-6-phosphate from GlcN-6-phosphate and acetyl coenzyme A. Early mercury-labelling experiments implicated a conserved cysteine in the reaction mechanism, whereas recent structural data appear to support a mechanism in which this cysteine plays no role. Here, two crystal structures of Caenorhabditis elegans GNA1 are reported, revealing an unusual covalent complex between this cysteine and the coenzyme A product. Mass-spectrometric and reduction studies showed that this inactive covalent complex can be reactivated through reduction, yet mutagenesis of the cysteine supports a previously reported bi-bi mechanism. The data unify the apparently contradictory earlier reports on the role of a cysteine in the GNA1 active site.