Accessing intractable, phosphorylated intrinsically disordered proteins via a protease‐cleavable inclusion body tag

Phosphorylated intrinsically disordered proteins (IDPs), such as Bcl2-associated agonist of cell death (BAD) and Tau, play critical roles in apoptosis and neurodegeneration, yet their instability during recombinant expression due to degradation, aggregation, and dephosphorylation, has limited structural and mechanistic studies. Here, we introduce the PINBody tag (PTM IDP Inclusion Body), a versatile inclusion body (IB)-targeting fusion that protects IDPs and their post-translational modifications during expression in Escherichia coli. When coupled with genetic code expansion (GCE), PINBody enables site-specific incorporation of phosphoserine (pS) while shielding this labile modification from dephosphorylation. In contrast to most IB-tag systems that rely on harsh chemical cleavage (e.g., acid, cyanogen bromide, heat) or slow, protease-based methods with limited refolding efficiency and IDP compatibility, PINBody supports rapid, complete protease-mediated tag removal under mild, refolding-compatible conditions. We demonstrate the generality of this system by producing milligram-scale quantities of phosphorylated mouse BAD (pS136), a protein that has evaded effective recombinant expression for over two decades, and 2N4R Tau phosphorylated at S404. To demonstrate PINbody utility, we generated homogeneous ¹⁵N-labeled Tau pS404 for 2D HSQC NMR analysis, which revealed chemical shift perturbations at and around S404 and confirmed that this phosphorylation primes GSK3β for site-specific modification at S400 and S396. This yielded a homogeneously triply phosphorylated Tau proteoform previously inaccessible by enzymatic means alone. Together, these results establish PINBody as a uniquely effective platform for producing native and site-specifically modified IDPs, enabling mechanistic dissection of phosphorylation cascades that were previously intractable to study.