Scalable One-Pot Production of Geranylgeranylated Proteins in Engineered Prokaryotes.

Abstract

Geranylgeranylation is a critical post-translational modification essential for various cellular functions. However, current methods for synthesizing geranylgeranylated proteins are complex and costly, which hinders access to these proteins for both biophysical and biomaterials applications. Here, we present a method for the one-pot production of geranylgeranylated proteins in Escherichia coli. We engineered E. coli to express geranylgeranyl pyrophosphate synthase (GGS), an enzyme that catalyzes the production of geranylgeranyl pyrophosphate. By coexpressing GGS with a geranylgeranyltransferase, we achieved efficient geranylgeranylation of model protein substrates, including intrinsically disordered elastin-like polypeptides (ELPs) and globular proteins such as mCherry and the small GTPases RhoA and Rap1B. We examined the biophysical behavior of the resulting geranylgeranylated proteins and observed that this modification affects the phase-separation and nanoassembly of ELPs and lipid bilayer engagement of mCherry. Taken together, our method offers a scalable, versatile, and cost-effective strategy for producing geranylgeranylated proteins, paving the way for advances in biochemical research, therapeutic development, and biomaterial engineering.