What question did this study set out to answer?

To develop an efficient expression system for recombinant L-asparaginase by optimizing RNA structure.

March 29, 2026Прикладная биохимия и микробиология / Applied Biochemistry and Microbiology0 citations

Optimization of RNA Structure Enhances Biosynthesis of L-Asparaginase from E. coli During Gene Overexpression

RSR. R. Shaifutdinov NON. A. Orlova IVI. I. Vorobyev

Key Points

To develop an efficient expression system for recombinant L-asparaginase by optimizing RNA structure.
Synthetic gene design for mRNA stability.
Use of E. coli BL21[DE3] strain for protein expression.
Shake-flask cultures for protein production evaluation.
Codon optimization to enhance translation efficiency.
Achieved 291 ± 9 mg/L of active L-asparaginase after 3 hours of induction.
Increased production was 50% higher than the control strain.
Improved mRNA secondary structure stability from –70 to –47 kcal/mol.
Purified enzyme met pharmacopeial standards and displayed high specific activity over 250 IU/mg.

Abstract

A highly efficient expression system for recombinant type II L-asparaginase from Escherichia coli (EC 3.5.1.1) was developed using a synthetic gene optimized for the folding energy of mRNA secondary structures in the 5-region. The engineered E. coli BL21DE3/pET28a-AsnSYN strain produced up to 291 ± 9 mg/L of enzymatically active protein (44.5 ± 2.6 mg/(L·OD)) in shake-flask cultures, 50% higher than the control strain with the native gene after 3 hours of induction. Codon optimization increased the mRNA secondary structure stability in the 5-region from –70 to –47 kcal/mol, potentially improving translation efficiency. The purified asparaginase met pharmacopeial standards, with a total yield ≥25%, specific activity 250 IU/mg, and high purity (no detectable impurities by electrophoresis,

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