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<p>Purification of active ribosomes is a prerequisite to studying ribosome functions. However, conventional purification methods such as ultracentrifugation, affinity tagging, and specialized chromatography present difficulties limiting their widespread use especially in laboratories with standard equipment. Here we describe a rapid and scalable method for purifying functionally active 70S ribosomes from <i>Escherichia coli</i> using strong anion exchange spin columns. Following optimization of buffer conditions for purification and precipitation steps for concentration, ribosomes can be prepared for cell-free protein synthesis in less than 1&#xa0;h using only standard laboratory resources. The approach is successfully applied to both small- and large-scale samples, recovering ribosomes with translational activity up to 70% of commercial standards and protein/RNA compositions comparable to established protocols. This versatile technique streamlines ribosome purification, enabling efficient access for studies in structural biology, antibiotic screening, and translational regulation, especially where resources or sample volumes are constrained.</p>

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Rapid and scalable purification of Escherichia coli active ribosomes using strong anion exchange spin columns

  • 期刊论文

  • 发表日期：

    2025年12月1日

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  • Ji Hyun Yu,
  • Hyegyung Kim,
  • Kathirvel Iyappan,
  • Joongoo Lee,
  • Hyungdon Yun,
  • Taek Jin Kang

摘要

Purification of active ribosomes is a prerequisite to studying ribosome functions. However, conventional purification methods such as ultracentrifugation, affinity tagging, and specialized chromatography present difficulties limiting their widespread use especially in laboratories with standard equipment. Here we describe a rapid and scalable method for purifying functionally active 70S ribosomes from Escherichia coli using strong anion exchange spin columns. Following optimization of buffer conditions for purification and precipitation steps for concentration, ribosomes can be prepared for cell-free protein synthesis in less than 1 h using only standard laboratory resources. The approach is successfully applied to both small- and large-scale samples, recovering ribosomes with translational activity up to 70% of commercial standards and protein/RNA compositions comparable to established protocols. This versatile technique streamlines ribosome purification, enabling efficient access for studies in structural biology, antibiotic screening, and translational regulation, especially where resources or sample volumes are constrained.