RT Journal Article
SR Electronic
T1 Saturation mutagenesis of a predicted ancestral Syk-family kinase
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2022.04.24.489292
DO 10.1101/2022.04.24.489292
A1 Helen T. Hobbs
A1 Neel H. Shah
A1 Sophie R. Shoemaker
A1 Jeanine F. Amacher
A1 Susan Marqusee
A1 John Kuriyan
YR 2022
UL http://biorxiv.org/content/early/2022/04/24/2022.04.24.489292.abstract
AB 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 Syk-family 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. Using this assay, we screened a site-saturation mutagenesis library of the kinase domain of this reconstructed Syk-family kinase. Sites of loss-of-function mutations identified in the screen correlate well with residues established previously as critical to function and/or structure in protein kinases. We also identified activating mutations in the regulatory hydrophobic spine and activation loop, which are within key motifs involved in kinase regulation. Strikingly, one mutation in an ancestral Syk-family variant increases the soluble expression of the protein by 75-fold. Thus, through ancestral sequence reconstruction followed by deep mutational scanning, we have generated Syk-family kinase variants that can be expressed in bacteria with very high yield.Competing Interest StatementThe authors have declared no competing interest.