Streamlining recombinant peptide and protein production using a pre-configured elastin-like polypeptide expression cassette
- 1Bridge Institute, Michelson Center for Convergent Bioscience, Structure Biology Center, University of Southern California, Los Angeles, California, United States of America
- 2Bridge Institute, Michelson Center for Convergent Bioscience, University of Southern California, Los Angeles, California, United States of America
Abstract
Elastin-like polypeptides (ELPs) are synthetic biopolymers that exhibit a unique thermo-responsive behavior: they remain soluble below a defined transition temperature but undergo reversible aggregation when the temperature exceeds this threshold. This property enables a simplified, non-chromatographic protein purification strategy. Despite these advantages, ELP-based purification has not yet been widely adopted as a standard method in research laboratories, indicating that certain practical limitations or challenges remain to be addressed. The first major challenge is the absence of a standardized ELP-tagged protein expression cassette suitable for use in research laboratories. The second is the development of an effective strategy for purifying the target protein released from the ELP tag following enzymatic cleavage. In this study, we present an optimized and modular gene cassette designed to standardize ELP-tagged peptide that can operates under mild conditions, featuring a low inverse transition temperature (Tt) of 27–31 °C, reduced salt concentrations (up to 0.5 M NaCl), and produces high yield and high purity product. Additionally, we exploited the ELP’s concentration induced aggregation to facilitate its separation from the target protein by filtration following enzymatic cleavage to address the second challenge. Here, we emphasize that this protocol has proven to be particularly efficient for the purification of small peptides, primarily due to the substantial size difference between the target peptide and the ELP tag. The purification of larger peptides remains feasible but may require alternative purification strategies. As a case study, we illustrate the purification of human β-defensin 1 (hBD-1; gene DEFB1), a 36-residue antimicrobial peptide, and discuss the challenges encountered and solutions implemented during the purification process.
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BibTeX
@article{lobos2026streamlining,
title = {{Streamlining recombinant peptide and protein production using a pre-configured elastin-like polypeptide expression cassette}},
author = {Jetson Lobos and Jeffrey Velasquez and Fariborz Nasertorabi},
journal = {PLOS One},
year = {2026},
month = sep,
doi = {10.1371/journal.pone.0357387},
url = {https://doi.org/10.1371/journal.pone.0357387},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Streamlining recombinant peptide and protein production using a pre-configured elastin-like polypeptide expression cassette
AU - Jetson Lobos
AU - Jeffrey Velasquez
AU - Fariborz Nasertorabi
PY - 2026
DA - 2026/09/11
JO - PLOS One
DO - 10.1371/journal.pone.0357387
UR - https://doi.org/10.1371/journal.pone.0357387
AB - Elastin-like polypeptides (ELPs) are synthetic biopolymers that exhibit a unique thermo-responsive behavior: they remain soluble below a defined transition temperature but undergo reversible aggregation when the temperature exceeds this threshold. This property enables a simplified, non-chromatographic protein purification strategy. Despite these advantages, ELP-based purification has not yet been widely adopted as a standard method in research laboratories, indicating that certain practical limitations or challenges remain to be addressed. The first major challenge is the absence of a standardized ELP-tagged protein expression cassette suitable for use in research laboratories. The second is the development of an effective strategy for purifying the target protein released from the ELP tag following enzymatic cleavage. In this study, we present an optimized and modular gene cassette designed to standardize ELP-tagged peptide that can operates under mild conditions, featuring a low inverse transition temperature (Tt) of 27–31 °C, reduced salt concentrations (up to 0.5 M NaCl), and produces high yield and high purity product. Additionally, we exploited the ELP’s concentration induced aggregation to facilitate its separation from the target protein by filtration following enzymatic cleavage to address the second challenge. Here, we emphasize that this protocol has proven to be particularly efficient for the purification of small peptides, primarily due to the substantial size difference between the target peptide and the ELP tag. The purification of larger peptides remains feasible but may require alternative purification strategies. As a case study, we illustrate the purification of human β-defensin 1 (hBD-1; gene DEFB1), a 36-residue antimicrobial peptide, and discuss the challenges encountered and solutions implemented during the purification process.
SN - 1932-6203
ER -