Engineering mRNA stability is a promising yet underexplored approach for improving recombinant protein production in bacterial systems. In this study, we evaluated the effect of synthetic 3'-UTR hairpin structures on mRNA stability and protein yield in Escherichia coli using two SUMO-fusion expression systems. Hairpin elements with defined structural features were introduced downstream of the coding sequence. In all constructs, 3'-UTR hairpins increased mRNA half-life, with stabilization ranging from approximately 2-fold to 3-fold (n = 3 biological replicates). In the SUMO-SARS-CoV-2-derived peptide system, enhanced transcript stability was accompanied by a marked increase in specific cellular fusion-protein content, reaching up to 6.8-fold relative to the control (n = 3). In the SUMO-liraglutide-derived peptide system, mRNA stabilization was also pronounced, and the increase in specific cellular fusion-protein content reached approximately 3-fold (p < 0.001, n = 6). These findings show that 3'-UTR engineering is an effective strategy for modulating mRNA stability in *E. coli*, but the quantitative relationship between transcript persistence and protein accumulation is context-dependent and likely influenced by additional factors, including translation efficiency. Overall, engineering of 3'-terminal RNA structures provides a practical tool for post-transcriptional tuning of recombinant expression systems.
Z. Khasanshina, M. Yarovikova, E. Buslaeva et al.· Protein Expression and Purif...· 0 citations
Incomplete removal of the initiator methionine is a frequent bottleneck in Escherichia coli-based production of recombinant proteins, causing heterogeneity and increased immunogenicity of biopharmaceuticals. Methionine aminopeptidase (MAP) is the key enzyme responsible for this post-translational modification, yet its endogenous activity is rapidly saturated under high-level expression conditions. Here, we report a case-study evaluation of a pBR322-derived expression system, previously applied to therapeutic peptides and insulin analogs, in combination with fed-batch cultivation for recombinant production of methionine aminopeptidase in E. coli. The map gene from E. coli BL21(DE3) was cloned into the pF644 vector to generate pF1492. During fed-batch cultivation under the tested conditions, specific productivity reached 127.03 ± 8.66 mg·g-1 and volumetric productivity of total cell-associated MAP reached 2.71 ± 0.18 g·L-1 by the final hour of induction. MAP accumulated predominantly as insoluble inclusion bodies, which is a common outcome for recombinant protein expression in E. coli at high rates. This study reports upstream production and inclusion body formation only; functional recovery and enzymatic activity were not assessed. Acetate remained moderate (35-60 mM) and biomass was stable, indicating balanced metabolism. The present study evaluates the performance of this system for methionine aminopeptidase as a stress-sensitive model protein under the tested conditions, without a side-by-side comparison with alternative expression systems.
G. Kuznetsov, Marina Yarovikova, E. Buslaeva et al.· Protein Expression and Purif...· 0 citations
