Biology subjects

Kunova, M. S.

Publications and source records attributed to Kunova, M. S..

3 recordsLinked to original sources

bioRxiv2025

Insights into the performance of CusF as a solubility tag for recombinant protein expression

The metal-binding periplasmic protein CusF has been proposed as a bifunctional tag enhancing solubility of recombinant proteins and enabling purification using Cu affinity chromatography. However, evidence for its performance remains limited to a few model proteins. Here, we evaluated CusF as a solubility tag for two heterologous proteins: a putative poly(A)-polymerase from Enterococcus faecalis (Efa PAP) and the red fluorescent protein mCherry. The proteins were fused to CusF, expressed in E. coli BL21 (DE3) pLysS and Rosetta 2 (DE3) strains, and assessed for solubility and IMAC binding. Native Efa PAP was completely insoluble under all tested conditions, and fusion to CusF did not improve its solubility. Similarly, CusF-mCherry accumulated predominantly in the in-soluble fraction, with only traces detectable in soluble lysates. Soluble CusF-mCherry did not bind Cu2+-charged IMAC resin, while moderate binding to Ni2+-charged resin was attributable to the vector-encoded His-tag rather than CusF. These results indicate that CusF does not universally enhance protein solubility and may not always bind Cu-based IMAC resin. Our findings expand empirical knowledge on solubility tag performance and emphasize the necessity of testing multiple tags to identify optimal strategies for recombinant protein production.

molecular biology↗

bioRxiv2025

The first poly(A) polymerase from Alphaproteobacteria

Bacterial poly(A) polymerases (PAPs) play important role in RNA metabolism but remain poorly characterized outside Gammaproteobacteria. Here, we cloned and biochemically characterized the first PAP from Alphaproteobacteria, specifically from Marinobacter lipolyticus (Mli PAP). Using homology-based screening against E. coli PAP-1, we identified Mli PAP, sharing 54.8% sequence identity with its E. coli counterpart. The enzyme was expressed in E. coli but formed insoluble inclusion bodies; active enzyme was solubilized and used for functional assays. Mli PAP exhibited optimal activity at 30{degrees}C and similar thermostability to E. coli PAP-1. ATP was the preferred substrate, with Km comparable to E. coli PAP-1 (1.61 mM and 1.70 mM, respectively), and Mg2+ (10 mM) was identified as the optimal cofactor. Mli PAP displayed salt-dependent activity, with the most effective polyadenylation in KCl and inhibition by NaCl and ammonium salts, contrasting with the halophilic nature of its host. This study provides the first functional insights into PAPs from Alphaproteobacteria, broadening the understanding of PAP diversity and biochemical properties, potential applications of PAPs in biotechnology.

molecular biology↗

bioRxiv2024

Optimization of conditions for production of soluble E. coli polyA-polymerase

Poly(A)-polymerase (PAP-1) from Escherichia coli is the primary enzyme responsible for synthesizing poly(A) tails on RNA molecules, signaling RNA degradation in bacterial cells. In vitro, PAP-1 is used to prepare libraries for RNAseq and to produce mRNA vaccines. However, E. coli PAP-1s toxicity and instability in low-salt buffers complicate its expression and purification. Here, we optimized the conditions for the production of recombinant PAP-1. For that, E. coli PAP-1 was expressed in seven E. coli strains with different origin and genetic backgrounds, followed by assessment of the overall protein yield, solubility, and enzymatic activity. Among the tested strains, BL21 (DE3) pLysS achieved the best balance of cell density, total PAP-1 yield, solubility, and specific activity. Rosetta 2 (DE3) and Rosetta Blue (DE3) hosting the pRARE plasmid exhibited the lowest solubility, likely due to excessive translation efficiency. Higher induction temperatures (>18{degrees}C) exacerbated PAP-1 insolubility. Interestingly, PAP-1 accumulation correlated with an increase in the plasmid copy number encoding the enzyme, indicating its potential utility as a surrogate marker for PAP-1 activity. These findings provide insights into optimizing E. coli PAP-1 production for biotechnological applications.

molecular biology↗