Advances on the large-scale bacterial production and stable phosphorylation of Hsc70.
Phosphorylation of the constitutively expressed Hsp70 chaperone Hsc70 at threonine 495 modulates its cellular functions. While the functional consequences of Hsc70 phosphorylation are increasingly understood, analyzing its structural effects is hampered by the need for large amounts of stably phosphorylated protein. Here, we establish a preparative protocol for producing phosphorylated Hsc70 (pHsc70) and its ATPase-deficient T204A (Hsc70T204A) mutant via the Legionella effector kinase LegK4, yielding 10mg protein per liter of cell culture. Hsc70 is expressed in Escherichia coli, purified by immobilized metal affinity chromatography, and enzymatically phosphorylated at T495 under conditions minimizing reverse reactions, achieving phosphorylation levels of ∼70% for Hsc70 and ∼86% for Hsc70T204A. Rigorous depletion of LegK4 following the phosphorylation reaction is critical, as residual kinase promotes dephosphorylation and compromises phosphorylation stability. Phosphorylation at T495 reduces the oligomerization propensity of Hsc70, as evidenced by SAXS measurements showing decreased radius of gyration, maximum particle dimension, and oligomer abundance in phosphorylated Hsc70T204A compared to the unphosphorylated protein across ATP-, ADP-, and apo-states. Thus, this protocol provides robust access to stably phosphorylated Hsc70 suitable for biochemical and structural investigations, and may offer a general strategy for preparative enzymatic phosphorylation using recombinant kinases.
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