Method for determining protein kinase substrate specificities by the phosphorylation of peptide libraries on beads, phosphate-specific staining, automated sorting, and sequencing.

A method is described for the elucidation of the peptide substrate phosphorylation specificity of a protein kinase. Peptide libraries with two to six degenerate positions and a length of seven or nine amino acids were generated directly on Sepharose beads by solid-phase peptide synthesis according to the split-and-mix procedure. The immobilized peptides were incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase (PKA) as a model enzyme resulting in the phosphorylation of the beads that contain the recognition motif of the kinase. The beads were then stained with a new phosphate-monoester-specific fluorescent dye consisting of a complex of iron(III) with fluorescein-coupled iminodiacetic acid. A flow cytometer was used to analyze the phosphorylation efficiency and the beads with the highest phosphorylation degree were isolated by the use of a fluorescence-activated cell sorter. Pool sequencing of those beads revealed the preferred kinase motif. The results are in good agreement with data from the literature. The method lends itself to the rapid elucidation of the specificity of uncharacterized protein kinases.

CD4 expressing human 293 cells as a tool for studies in HIV-1 replication: the efficiency of translational frameshifting is not altered by HIV-1 infection.

An adherent human cell line (293) was made susceptible for HIV-1 infection by transfer of a CD4 expression plasmid. These cells could be infected with HIV-1 and produced infectious virus up to a titer of 10(6) TCID50/ml releasing p24 protein up to 1 micrograms/ml. Since they can be efficiently transfected with reporter genes, these cells are a suitable model system to monitor biochemical events during productive infection of HIV-1 and can be used for antiviral drugs. Translational frameshifting determines the balance of the structural Gag versus the catalytic Pol proteins which is probably crucial for correct virus assembly. We have genetically engineered CD4 expressing 293 cells with a sensitive in vivo reporter system to monitor the extent of frameshifting in HIV-1-infected versus uninfected cells. During the time course of productive HIV-1 infection the low efficiency of ribosomal frameshifting is not altered.