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Background <p>The homogeneity and authenticity of recombinant proteins are crucial factors that directly impact the quality of biopharmaceutical products. These attributes ensure the consistency of therapeutic efficacy in protein-based drugs and contribute to their stability and safety. However, obtaining homogeneous and authentic recombinant proteins remains a considerable challenge. For cytoplasmic expression this includes the complexities associated with potential heterogeneity of N-terminal methionine processing. This study aimed to overcome that limitation by introducing an innovative in vivo cleavage system that is capable of producing a wide range of recombinant proteins with homogeneous and authentic N-termini.</p> Results <p>Our in vivo cleavage strategy involves the coexpression of a proline iminopeptidase from <i>Hafnia alvei</i> (PIPHA) alongside the protein of interest (POI) with an additional proline inserted after the initiating methionine. Methionine amino peptidase efficiently removes the initiating methionine and subsequently, the proline is removed by PIPHA. This enables the generation of recombinant proteins with homogeneous and authentic N-termini. The P1’ P2’ substrate specificity of PIPHA suggested that the system can be used for a wide range of proteins, and our results indicate it can be used across different host strains and media. PIPHA activity was enhanced by targeting the pre-folded state of proteins using pre-expression and ribosome association strategies, which increased the efficiency of the system up to 100%.</p> Conclusion <p>This study created a pioneering in vivo cleavage system with remarkable versatility, which might be applied in the production of high-quality homogenous proteins. By opting for in vivo rather than in vitro strategies, our system is expected to provide a more sustainable and environmentally friendly alternative. Further investigations aimed at optimizing system efficiency and scaling production will evaluate its applicability in pharmaceutical protein manufacturing.</p>

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Proline iminopeptidase: a novel in vivo strategy to produce proteins with homogeneous and authentic N-termini in E. coli

  • 期刊论文

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  • 发表日期：

    2026年8月4日

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  • Thu Pham Anh Nguyen,
  • Zinke Vanderdeelen,
  • Lukas Rettenbacher,
  • Mirva J. Saaranen,
  • Lloyd W. Ruddock

摘要

Background

The homogeneity and authenticity of recombinant proteins are crucial factors that directly impact the quality of biopharmaceutical products. These attributes ensure the consistency of therapeutic efficacy in protein-based drugs and contribute to their stability and safety. However, obtaining homogeneous and authentic recombinant proteins remains a considerable challenge. For cytoplasmic expression this includes the complexities associated with potential heterogeneity of N-terminal methionine processing. This study aimed to overcome that limitation by introducing an innovative in vivo cleavage system that is capable of producing a wide range of recombinant proteins with homogeneous and authentic N-termini.

Results

Our in vivo cleavage strategy involves the coexpression of a proline iminopeptidase from Hafnia alvei (PIPHA) alongside the protein of interest (POI) with an additional proline inserted after the initiating methionine. Methionine amino peptidase efficiently removes the initiating methionine and subsequently, the proline is removed by PIPHA. This enables the generation of recombinant proteins with homogeneous and authentic N-termini. The P1’ P2’ substrate specificity of PIPHA suggested that the system can be used for a wide range of proteins, and our results indicate it can be used across different host strains and media. PIPHA activity was enhanced by targeting the pre-folded state of proteins using pre-expression and ribosome association strategies, which increased the efficiency of the system up to 100%.

Conclusion

This study created a pioneering in vivo cleavage system with remarkable versatility, which might be applied in the production of high-quality homogenous proteins. By opting for in vivo rather than in vitro strategies, our system is expected to provide a more sustainable and environmentally friendly alternative. Further investigations aimed at optimizing system efficiency and scaling production will evaluate its applicability in pharmaceutical protein manufacturing.