(666b) Engineering Tyrosine Sulfation in E. coli

Posttranslational modifications are known to regulate the activity and solubility of proteins. Tyrosine sulfation plays a critical role in mediating protein-protein interactions in multicellular eukaryotes. For example, the interaction between tyrosine sulfated chemokine receptor 5 (CCR5) and HIV viral coat protein, gp120, is dependent on tyrosine sulfation. Furthermore, the tyrosine sulfate mediated interactions are highly specific, even when compared to phosphorylated and native tyrosine analogs. Human antibodies against HIV are known to employ CCR5 mimicry through the inclusion of a sulfated tyrosine in the CDR3 loop. Inspired by this example, we wish to develop a protein-engineering platform in E. coli that utilizes tyrosine sulfation in its products. As nearly all single cell organisms are not known to have tyrosyl protein sulfotransferases (TPSTs), the first step forward is to establish a tyrosine sulfation pathway in E. coli. We use a recently discovered TPST from a gram-negative bacterium to enable tyrosine sulfation in E. coli. This enzyme is expressed as a soluble cytoplasmic protein that utilizes PAPS for a sulfate donor group. The endogenous PAPS generated by E. coli was initially used and further enhanced by over-expression. We demonstrate the efficiency of this synthetic tyrosine sulfation approach through co-expression of bacterial TPSTs with known tyrosine sulfation target peptides fused to maltose binding protein and inserted into variable heavy chain Ig. Our results enable further engineering of high affinity specific protein-ligand interactions utilizing tyrosine sulfation.