Fluorescence imaging is perhaps the most powerful technique currently available for continuously observing the dynamic intracellular biochemistry of single living cells. However, fluorescent indicator dyes have been available only for simple inorganic ions such as Ca2+, H+, Na+, K+, Mg2+ and Cl-. We now report a fluorescent indicator for the adenosine 3',5'-cyclic monophosphate (cAMP) signalling pathway. The sensor consists of cAMP-dependent protein kinase in which the catalytic (C) and regulatory (R) subunits are each labelled with a different fluorescent dye such as fluorescein or rhodamine capable of fluorescence resonance energy transfer in the holoenzyme complex R2C2. When cAMP molecules bind to the R subunits, the C subunits dissociate, thereby eliminating energy transfer. The change in shape of the fluorescence emission spectrum allows cAMP concentrations and the activation of the kinase to be nondestructively visualized in single living cells microinjected with the labelled holoenzyme.

The ribosomal incorporation of nonnative amino acids into polypeptides in living cells provides the opportunity to endow therapeutic proteins with unique pharmacological properties. We report here the first clinical study of a biosynthetic protein produced using an expanded genetic code. Incorporation of p-acetylphenylalanine (pAcF) at distinct locations in human growth hormone (hGH) allowed site-specific conjugation with polyethylene glycol (PEG) to produce homogeneous hGH variants. A mono-PEGylated mutant hGH modified at residue 35 demonstrated favorable pharmacodynamic properties in GH-deficient rats. Clinical studies in GH-deficient adults demonstrated efficacy and safety comparable to native human growth hormone therapy but with increased potency and reduced injection frequency. This example illustrates the utility of nonnative amino acids to optimize protein therapeutics in an analogous fashion to the use of medicinal chemistry to optimize conventional natural products, low molecular weight drugs, and peptides.protein engineering | endocrinology | bio-better

Ligand-mediated targeting of DNA was validated by condensing a plasmid DNA encoding the ␤-galactosidase (␤-gal) gene with a basic fibroblast growth factor (FGF2) that was first chemically conjugated to polylysine (K). The conditions that gave optimal binding of this FGF2 to DNA also generated the highest level of ␤-gal expression when added to FGF2 target cells like COS-1, 3T3, baby hamster kidney (BHK), or endothelial cells. This ␤-gal activity increased in a time-and dose-dependent manner and was dependent on the inclusion of FGF2 in the complex. FGF receptor specificity was demonstrated by competition of the complex with FGF2 and heparin, and by the failure of cytochrome c or histone H1 to mimic the gene-targeting effects of FGF2. The expression of ␤-gal was also endosome dependent because chloroquine increased ␤-gal expression 8-fold and endosome disruptive peptides increased expression of ␤-gal 26-fold. Taken together these findings establish that DNA can be introduced into cells through the high affinity FGF receptor complex, and while its efficiency will require significant enhancements to achieve sustained and elevated transgene expression, the possibility that the technique could be used to deliver DNAs encoding cytotoxic molecules is discussed.

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