| Epidermal growth factor receptor, the primary member of the tyrosine kinase family of signaling cell surface receptors, and asparagine synthetase, the eukaryotic enzyme responsible for the synthesis of asparagine from ATP, glutamine and aspartate, have been the focus of intense structural, mechanistic and inhibitory studies, as they represent two molecular targets for anti-cancer drug discovery. Human asparagine synthetase and five of its mutants were cloned and expressed in a baculovirus expression system under the control of the very late polyhedrin promoter. The recombinant proteins fused to a C-terminal histidine tag were purified to homogeneity in a single-step, nickel-chelation chromatography and the apparent affinity constants were determined. The wild-type asparagine synthetase was properly processed, as N-terminus sequence analysis showed that the starting methionine residue was cleaved off. The recombinant enzyme had both glutaminase and glutamine-dependent asparagine synthetase activities. As expected, Cys-1 mutants showed no glutamine-dependent activity, but were able to use ammonia as a nitrogen source for asparagine synthesis. Mutation of the conserved position Arg-339 to alanine or lysine completely eliminated the synthetase activity, while preserving the ability to hydrolyze glutamine. A possible thermolabile mutant, Lys413-Phe, showed no activity even at lower temperatures. The glutaminase and glutamine-dependent asparagine synthetase activities required chloride ions, which could be replaced by bromide or iodide, but with less activity. Compounds analogous to the reaction transition state or the β-aspartyl-AMP intermediate were effective inhibitors of the synthetase activity, but did not interfere with the glutaminase activity of the recombinant enzyme.; The extracellular domain of epidermal growth factor receptor was expressed in insect cells with a baculovirus secretion system and purified from the serum-free medium based on a C-terminal histidine tag. The domain was biotinylated and used as a target to select 12-residue peptides displayed on bacteriophage from a 1.9 × 109-member library. After three rounds of affinity selection, binding phage were purified and clones were analyzed to identify the sequences of selected peptides. Analysis of the amino-acid sequences of selected peptides showed a high content of histidine residues, indicating that selection was directed against Ni2+ ions that are possibly contaminating the target by binding to the C-terminal tag. Two peptides were synthesized and evaluated. While both peptides were able to displace TGFα bound to the epidermal growth factor receptor, only the peptide with no histidines had an antagonistic activity against growth factor-induced cell proliferation. |
