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Title: Saturation mutagenesis of a predicted ancestral Syk-family kinase
Authors: Hobbs, Helen T.
Keywords: Đột biến; kinase; bão hòa; Syk
Issue Date: 2023
Publisher: bioRxiv
Abstract: Many tyrosine kinases cannot be expressed readily in E. coli, limiting facile production of these proteins for biochemical experiments. We used ancestral sequence reconstruction to generate a spleen tyrosine kinase (Syk) variant that can be expressed in bacteria and purified in soluble form, unlike the human members of this family (Syk and ZAP-70). The catalytic activity, substrate specificity, and regulation by phosphorylation of this Syk variant are similar to the corresponding properties of human Syk and ZAP-70. Taking advantage of the ability to express this novel Sykfamily kinase in bacteria, we developed a two-hybrid assay that couples the growth of E.coli in the presence of an antibiotic to successful phosphorylation of a bait peptide by the kinase.
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