The effect of protein acetylation on the formation and processing of inclusion bodies and endogenous protein aggregates in Escherichia coli cells

Abstract

BackgroundAcetylation of lysine residues is a reversible post-translational modification conserved from bacteria to humans. Several recent studies have revealed hundreds of lysine-acetylated proteins in various bacteria; however, the physiological role of these modifications remains largely unknown. Since lysine acetylation changes the size and charge of proteins and thereby may affect their conformation, we assumed that lysine acetylation can stimulate aggregation of proteins, especially for overproduced recombinant proteins that form inclusion bodies.ResultsTo verify this assumption, we used Escherichia coli strains that overproduce aggregation-prone VP1GFP protein. We found that in ΔackA-pta cells, which display diminished protein acetylation, inclusion bodies were formed with a delay and processed faster than in the wild-type cells. Moreover, in ΔackA-pta cells, inclusion bodies exhibited significantly increased specific GFP fluorescence. In CobB deacetylase-deficient cells, in which protein acetylation was enhanced, the formation of inclusion bodies was increased and their processing was significantly inhibited. Similar results were obtained with regard to endogenous protein aggregates formed during the late stationary phase in ΔackA-pta and ΔcobB cells.ConclusionsOur studies revealed that protein acetylation affected the aggregation of endogenous E. coli proteins and the yield, solubility, and biological activity of a model recombinant protein. In general, decreased lysine acetylation inhibited the formation of protein aggregates, whereas increased lysine acetylation stabilized protein aggregates. These findings should be considered during the designing of efficient strategies for the production of recombinant proteins in E. coli cells.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0590-8) contains supplementary material, which is available to authorized users.

Trehalose protects Escherichia coli against carbon stress manifested by protein acetylation and aggregation

“…These results are in agreement with the in vitro experiment (Fig. 5) and with our previous studies showing that in the stationary-phase ΔackA-pta cells with decreased Nε-lysine acetylation, protein aggregation was diminished (Kuczyńska-Wiśnik et al, 2016). It should be noted, however, that there are reported cases of protein aggregation induced by acetylation (Cohen et al, 2011;Olzscha et al, 2017) and stabilization by acetylation (Li et al, 2010).…”

“…To further test our hypothesis, the influence of the Δ otsA mutation on the formation of inclusion bodies (IBs) in cells overproducing a fusion protein VP1GFP was investigated (García‐Fruitós et al , ; Kuczyńska‐Wiśnik et al , ). Because VP1GFP retained its fluorescence in inclusion bodies, it was possible to examine the size of aggregates by fluorescence microscopy.…”

“…To prepare whole cell extracts for in vitro acetylation, Δ ackA‐pta [pVP1GFP] cells were pelleted, converted into spheroplasts and lysed by sonication as described previously (Kuczyńska‐Wiśnik et al , ). For AcP treatment of proteins, whole cell extracts were incubated with freshly prepared 10 mM of lithium potassium acetyl phosphate (Sigma) for 16 h at 37°C (Weinert et al , , with modifications).…”

Mutli-Features Prediction of Protein Translational Modification Sites

“…All the above mentioned protein segements and polypeptides sequences have been derived from the UniProt, which is the well-known protein database in the field of bioinformatics [16]. It has been utilized in studying and researching enzyme specificity (ES) [17] as well as protein-protein binding sites (PPB) [23][24].…”

“…All the above mentioned protein segements and polypeptides sequences have been derived from the UniProt, which is the well-known protein database in the field of bioinformatics [16]. It has been utilized in studying and researching enzyme specificity (ES) [17], signal peptide/ amino acid residues' cleavage sites (AACS) [18], hydroxyproline and hydroxylysine sites (H2S) [19] methylation sites [20], nitrotyrosine sites (NiS) [21], protein-protein interaction (PPI) [22], and protein-protein binding sites (PPB) [23][24].…”