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Updated: Aug 5, 2026
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Detection and Removal of Nuclease Contamination During Purification of Recombinant Prototype Foamy Virus Integrase
Published on: December 8, 2017
Simultaneous solubility-enhancing domain removal and target purification using the Npu split intein
Sophia Mayone1, Shanthi Napier1, Roman Adomanis1
1The Ohio State University William G Lowrie Department of Chemical and Biomolecular Engineering, 151 West Woodruff Ave., Columbus, OH, 43210, USA.
Protein Expression and Purification
|August 3, 2026
Summary
This study introduces a novel split intein system for simultaneous purification and solubility-enhancing domain removal of challenging recombinant proteins, offering a streamlined downstream process for biologics. The method yields highly pure and active therapeutic proteins.
Area of Science:
- Biotechnology
- Protein Engineering
- Biochemistry
Background:
- Current antibody purification methods like Protein A are not universally applicable to non-antibody therapeutics.
- Recombinant protein expression in E. coli often requires solubility-enhancing domains (SEDs), complicating downstream processing due to necessary cleavage steps.
Purpose of the Study:
- To demonstrate the efficacy of the Nostoc punctiforme (Npu) DnaE split intein platform for simultaneous purification and SED removal of challenging target proteins.
- To evaluate the platform's performance with various therapeutic proteins, including human fibroblast growth factor-2, interleukin-2, and an anti-CTLA-4 nanobody.
Main Methods:
- Utilized N-terminal fusion of SEDs with the Npu DnaE split intein tag to target proteins for enhanced soluble expression.
- Employed ÄKTA FPLC purification and characterization assays to assess protein purity, solubility, and DNA clearance.
- Introduced a single-point mutation in the intein tag to optimize cleavage rates for difficult targets.
Main Results:
- Achieved at least 75% solubility for target proteins (FGF-2, IL-2, nCTLA-4) when fused with SEDs.
- Obtained up to 98% purity and 98% DNA clearance using a single intein column purification.
- Demonstrated biological activity of purified FGF-2 and nCTLA-4 through activity assays.
Conclusions:
- The Npu DnaE split intein platform offers a streamlined and efficient method for downstream processing of SED-dependent proteins.
- This intein-mediated purification strategy shows significant potential for therapeutic protein production beyond the discovery phase.
