One-Step Purification of Multi-Subunit RNA Polymerases Using CL7/Im7

Original Scientific Article Published: April 2020

Multi-subunit proteins, such as bacterial RNA polymerases, remain technically challenging targets in structural biology and assay development. Their coordinated expression, complex assembly, and sensitivity to purification conditions introduce significant variability—often requiring 3–5 chromatography steps to yield usable material.

TriAltus BioScience addressed these challenges by applying our CL7/Im7 one-step purification system, integrated with custom vector design, to streamline production of T. thermophilus and M. tuberculosis RNA polymerases.

Using a single T7 promoter to drive expression of all subunits in a monocistronic arrangement, we promoted synchronous translation and in vivo assembly of each polymerase complex. A CL7 affinity tag was strategically fused to the N-terminal of the beta-prime subunit, enabling precise immobilization on Im7 resin.

Key Outcomes

  • >99% Target Protein Purity: Verified by SDS-PAGE and silver stain, our preparations demonstrated near-total exclusion of background contaminants. This purity threshold reduces noise in downstream applications such as enzyme kinetics, nucleic acid interaction assays, and crystallographic screens.

  • Coordinated Multi-Subunit Expression: Unlike co-transfection or multi-vector systems, our monocistronic vector strategy minimizes stoichiometric imbalance, improving the fidelity of complex assembly and reducing the risk of truncation or misfolded intermediates.

  • Enhanced Recovery Yield: By enabling a true one-step capture-and-release process, we recovered more assembled, active enzyme while eliminating the need for polishing steps like ion exchange or SEC. This improves throughput and reproducibility in protein core facilities.

  • Streamlined Workflow: Im7-based affinity purification, followed by on-resin PreScission protease cleavage, shortened the purification window and limited protease exposure, preserving structural integrity and enzymatic activity.

 

Ideal Applications

  • Researchers in structural biology needing large quantities of homogenous, stable complexes for X-ray or cryo-EM studies

  • Scientists working in transcriptional regulation or CRISPR tool development, where enzyme function and multimeric architecture must be preserved

  • Biotech teams developing assays using RNA polymerase or other RNP targets, where background protein interference must be minimized

TriAltus supports teams working at the cutting edge of RNA biology, synthetic enzymology, and structural research. Our proprietary purification technology allows researchers to focus on insights—not infrastructure. If your research team is interested in a demo of our technology, schedule an appointment with us!

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