Department of Chemistry, University of California, Berkeley, California, USA.
Department of Biomedical Engineering, University of California, Irvine, California, USA.
Protein Sci. 2022 Oct;31(10):e4411. doi: 10.1002/pro.4411.
Many tyrosine kinases cannot be expressed readily in Escherichia 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 zeta-chain-associated protein kinase of 70 kDa [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.
许多酪氨酸激酶在大肠杆菌中不易表达，这限制了这些蛋白质在生化实验中的轻易生产。我们使用祖先序列重建生成了一种脾酪氨酸激酶（Syk）变体，可以在细菌中表达并以可溶性形式纯化，这与该家族的人类成员（Syk 和 70kDa 链关联蛋白激酶[ZAP-70]）不同。这种 Syk 变体的催化活性、底物特异性和磷酸化调节与人类 Syk 和 ZAP-70 的相应特性相似。利用在细菌中表达这种新型 Syk 家族激酶的能力，我们开发了一种双杂交测定法，该测定法将大肠杆菌在抗生素存在下的生长与激酶成功磷酸化诱饵肽偶联起来。使用该测定法，我们筛选了该重建的 Syk 家族激酶的激酶结构域的饱和点突变文库。筛选中确定的无功能突变位点与先前确定的对蛋白激酶的功能和/或结构至关重要的残基很好地相关。我们还在调节疏水性脊和激活环中鉴定出激活突变，这些突变位于与激酶调节相关的关键基序内。引人注目的是，祖先 Syk 家族变体中的一个突变将蛋白质的可溶性表达提高了 75 倍。因此，通过祖先序列重建和深度突变扫描，我们生成了可在细菌中高产量表达的 Syk 家族激酶变体。
