engleski

Protein tyrosine phosphorylation in Streptomycetes

We previously showed evidence for protein phosphorylation in a number of Streptomyces species. The patterns of tyrosine phosphorylations varied during the growth phase and were also influenced by culture conditions. We report here the results of two different strategies employed for the further analysis of tyrosine phosphorylated proteins. One approach is based on the comprehensive process for the protein isolation. Enrichment of the phosphoproteins after ammonium sulfate precipitation was obtained by antiphosphotyrosine affinity chromatography. Eluted proteins were applied to a MonoQ column. The peptides in the concentrated and the active fraction were resolved by preparative SDS-PAGE . After trypsin digestion several peptides were sequenced by nanoelectrospray mass spectrometry. On the basis of this, PCR primers were design and sequencing of one amplified DNA region is currently underway. The second approach involved Western blott analysis with antiphospho-Stat3 and anti-Stat3. Using this method, a protein with similarity to a eukaryotic STAT was found. With PCR primers designed against critical motifs in STAT family members we were able to amplify a region that shows homology to the STAT DNA binding domain and the STAT-SH2 domain. This region also shows homology to the SH3 domain from Abl kinase.

nije evidentirano

nije evidentirano