A Facile Protocol to Generate Site-Specifically Acetylated Proteins in <em>Escherichia Coli</em>

Abstract

Post-translational modifications that occur at specific positions of proteins have been shown to play important roles in a variety of cellular processes. Among them, reversible lysine acetylation is one of the most widely distributed in all domains of life. Although numerous mass spectrometry-based acetylome studies have been performed, further characterization of these putative acetylation targets has been limited. One possible reason is that it is difficult to generate purely acetylated proteins at desired positions by most classic biochemical approaches. To overcome this challenge, the genetic code expansion technique has been applied to use the pair of an engineered pyrrolysyl-tRNA synthetase variant, and its cognate tRNA from Methanosarcinaceae species, to direct the cotranslational incorporation of acetyllysine at the specific site in the protein of interest. After first application in the study of histone acetylation, this approach has facilitated acetylation studies on a variety of proteins. In this work, we demonstrated a facile protocol to produce site-specifically acetylated proteins by using the model bacterium Escherichia coli as the host. Malate dehydrogenase was used as a demonstration example in this work.

Characterizing lysine acetylation of Escherichia coli type II citrate synthase

“…Interestingly, acetylation at K283 increased the activity by nearly twofold, while acetylation at K295 decreased the activity by about 10‐fold. Since acetylation could occur at multiple lysine residues in the same protein, we also generated a dual‐acetylated variant at K283 and K295 simultaneously by the genetic code expansion strategy , which still had a significant activity loss ( P < 0.0001), implying the key role of acetylation at K295 in regulating CS enzyme activities. To exclude the possibility that the decreased activity was caused by mis‐folding of the acetylated variant, we performed circular dichroism (CD) analysis of the CS‐295AcK variant.…”

“…Wild‐type CS and its variants were expressed from the pCDF‐1b plasmid (EMD Millipore, Burlington, MA, USA) with a C‐terminal His 6 ‐tag in BL21 (DE3) cells (New England Biolabs). The protocols of purification were described in previous studies with slightly modifications . The expression strain was grown in LB medium.…”

Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli

“…Then we applied the genetic code expansion approach to incorporate acetyllysine directly into those selected positions by our recently optimized protocol individually [54]. This approach utilizes an engineered Methanosarcina barkeri pyrrolysyl-tRNA synthetase [55] and an optimized cognate tRNA pyl [56] to read through the TAG stop codon in the gene of ICDH introduced by site-directed mutagenesis as acetyllysine, thus producing homogeneously acetylated ICDH variants at selected sites [57, 58].…”

A Synthetic Reporter for Probing Mistranslation in Living Cells

“…Detailed procedures were in the Supplementary Material . The site-specifically acetylated ThrRS variant was generated by our established genetic incorporation system ( Venkat et al, 2017a ). Protein concentrations were measured by the Bradford Protein Assay (Bio-Rad, Hercules, CA, United States).…”