Soluble recombinant CD69 receptors optimized to have an exceptional physical and chemical stability display prolonged circulation and remain intact in the blood of mice.

We investigated the soluble forms of the earliest activation antigen of human leukocyte CD69. This receptor is expressed at the cell surface as a type II homodimeric membrane protein. However, the elements necessary to prepare the soluble recombinant CD69 suitable for structural studies are a matter of controversy. We describe the physical, biochemical and in vivo characteristics of a highly stable soluble form of CD69 obtained by bacterial expression of an appropriate extracellular segment of this protein. Our construct has been derived from one used for CD69 crystallization by further optimization with regard to protein stability, solubility and easy crystallization under conditions promoting ligand binding. The resulting protein is stable at acidic pH and at temperatures of up to 65 degrees C, as revealed by long-term stability tests and thermal denaturation experiments. Protein NMR and crystallography confirmed the expected protein fold, and revealed additional details of the protein characteristics in solution. The soluble CD69 refolded in a form of noncovalent dimers, as revealed by gel filtration, sedimentation velocity measurements, NMR and dynamic light scattering. The soluble CD69 proved to be remarkably stable in vivo when injected into the bloodstream of experimental mice. More than 70% of the most stable CD69 proteins is preserved intact in the blood 24 h after injection, whereas the less stable CD69 variants are rapidly taken up by the liver.

Overexpression and purification of Pyrococcus abyssi phosphopantetheine adenylyltransferase from an optimized synthetic gene for NMR studies.

Phosphopantetheine adenylyltransferase (PPAT) is an essential enzyme that catalyses a rate-limiting step in coenzyme A (CoA) biosynthesis in all organisms. This study was conducted to obtain a high amount of pure, soluble, and stable PPAT from the hyperthermophilic archaeon Pyrococcus abyssi with the aim of investigating its structural characterization by NMR. Production of this enzyme from its natural gene in the Escherichia coli classical expression strain (BL21(DE3)) was not possible, most likely due to the presence of a high number of E. coli rare codons. Only a low amount of P. abyssi PPAT was previously obtained in two E. coli strains encoding tRNAs that recognize these rare E. coli codons and only by using a very rich growth medium. It was not possible to use this strategy to prepare labelled samples for the NMR study, thus another solution had to be found. Therefore, a synthetic gene encoding P. abyssi PPAT was constructed for which not only the rare codons were changed but which was also optimized to avoid other expression-limiting factors such as internal ribosome entry sites, RNA secondary structures, and DNA repeats. Gene optimization strongly increased the yield of P. abyssi PPAT in E. coli BL21(DE3) and allowed us to start the structural characterization of the enzyme. Circular dichroism and 2D NMR experiments indicate the presence of a well-ordered structure for P. abyssi PPAT and also confirm the existence of this enzyme as a monomer in solution.