Characterization of S-thiolation on secreted proteins from E. coli by mass spectrometry.

S-thiolation is a reversible post-translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells. Since it can alter protein functions and introduce molecular heterogeneity, S-thiolation is undesirable for recombinant protein production. To date, few published studies have characterized thiol modifiers or investigated the mechanism of S-thiolation in recombinant proteins. In this work, reversed-phase liquid chromatography and mass spectrometry were used to characterize four of the most abundant thiol modifiers on recombinant proteins secreted from E. coli BL21 (DE3) strain. These thiol modifiers have been identified as glutathione, 4-phosphopantetheine, gluconoylated glutathione, and dephosphorylated coenzyme A. S-thiolation by these thiol modifiers increases protein mass by 305, 356, 483, and 685 Da, respectively. These specific mass increases can be used as markers for identifying S-thiolation in recombinant proteins.

Synthesis of orthogonally reactive FK506 derivatives via olefin cross metathesis.

Chemical inducers of dimerization (CIDs) are employed in a wide range of biological applications to control protein localization, modulate protein-protein interactions and improve drug lifetimes. These bifunctional chemical probes are assembled from two synthetic modules, which each provide affinity for a distinct protein target. FK506 and its derivatives are often employed as modules in the syntheses of these bifunctional constructs, owing to the abundance and favorable distribution of their target, FK506-binding protein (FKBP). However, the structural complexity of FK506 necessitates multi-step syntheses and/or multiple protection-deprotection schemes prior to installation into CIDs. In this work, we describe an efficient, one-step synthesis of FK506 derivatives through a selective, microwave-accelerated, cross metathesis diversification step of the C39 terminal alkene. Using this approach, FK506 is modified with an array of functional groups, including primary amines and carboxylic acids, which make the resulting derivatives suitable for the modular assembly of CIDs. To illustrate this idea, we report the synthesis of a heterobifunctional HIV protease inhibitor.