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Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins.

Daniel Bello-Gil1, Beatriz Maestro1, Jennifer Fonseca1

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The BioF affinity tag enables robust protein immobilization onto polyhydroxyalkanoates (PHAs), creating versatile bioactive materials. This method offers stable protein binding and tunable adsorption for advanced biomaterial engineering.

Keywords:
PHBaffinity tagphasinspolyhydroxyalkanoatesprotein immobilization

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Area of Science:

  • Biotechnology and Biomaterials Engineering
  • Polymer Science and Engineering
  • Protein Engineering and Bioconjugation

Background:

  • Polyhydroxyalkanoates (PHAs) are biodegradable polyesters produced by bacteria, with potential applications in biomaterial development.
  • Protein immobilization onto biopolymers is crucial for creating functional biomaterials, but efficient and stable methods are needed.

Purpose of the Study:

  • To investigate the PHA-binding domain of Pseudomonas putida KT2440 PhaF phasin (BioF) as an affinity tag for in vitro protein functionalization of poly-3-hydroxybutyrate (PHB) particles.
  • To evaluate the stability and tunability of protein-biopolyester interactions mediated by the BioF tag.
  • To demonstrate the utility of BioF-functionalized PHAs in a minibioreactor for stable enzymatic activity.

Main Methods:

  • Recombinant proteins, including full-length PhaF and BioF fusion proteins (BioF-C-LytA, BioF-β-galactosidase), were expressed and purified.
  • In vitro immobilization of these proteins onto poly-3-hydroxybutyrate (PHB) particles using the BioF affinity tag.
  • Assessment of protein-biopolyester interaction stability across various pH and temperature conditions.
  • Modulation of binding strength using amphiphilic compounds and evaluation of immobilized enzyme activity in a minibioreactor.

Main Results:

  • The BioF tag facilitated strong and stable in vitro immobilization of recombinant proteins onto PHB particles.
  • Protein-PHA interactions remained stable over a broad range of pH and temperatures, with bound proteins protected from degradation.
  • Binding strength was tunable via surface coating, and BioF-β-galactosidase exhibited stable enzymatic activity after repeated cycles in a minibioreactor.

Conclusions:

  • The BioF affinity tag is a versatile tool for functionalizing PHA supports with recombinant proteins, enabling the creation of novel bioactive materials.
  • The broad applicability of the BioF tag to various PHAs and its tunable binding properties offer significant potential for biomaterial engineering.
  • This approach provides new perspectives for developing advanced bioactive materials requiring controlled protein loading and enhanced stability.