Dissecting the Polyhydroxyalkanoate-Binding Domain of the PhaF Phasin: Rational Design of a Minimized Affinity Tag

Abstract

Phasin PhaF from Pseudomonas putida consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for binding to the polyhydroxyalkanoate (PHA) granule. BioF has been exploited for biotechnological purposes as an affinity tag in the functionalization of PHA beads with fusion proteins both in vivo and in vitro. The structural model of this domain suggests an amphipathic α-helical conformation with the hydrophobic residues facing the PHA granule. In this work, we analyzed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for in vivo localization on PHA, stability on the surface of the PHA granule against pH, temperature, and ionic strength, and their possible influence on PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP) that retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing the functionality and stability of the fusion proteins MinP–β-galactosidase and MinP-CueO from Escherichia coli. IMPORTANCE Polyhydroxyalkanoates (PHAs) are biocompatible, nontoxic, and biodegradable biopolymers with exceptional applications in the industrial and medical fields. The complex structure of the PHA granule can be exploited as a toolbox to display molecules of interest on their surface. Phasins, the most abundant group of proteins on the granule, have been employed as anchoring tags to obtain functionalized PHA beads for high-affinity bioseparation, enzyme immobilization, diagnostics, or cell targeting. Here, a shorter module based on the previously designed BioF tag has been demonstrated to maintain the affinity for the PHA granule, with higher stability and similar functionalization efficiency. The use of a 67% shorter peptide, which maintains the binding properties of the entire protein, constitutes an advantage for the immobilization of recombinant proteins on the PHA surface both in vitro and in vivo.

Enzybiotic-mediated antimicrobial functionalization of polyhydroxyalkanoate nanoparticles

“…However, a concentration-dependent elution was observed for Cpl-711 upon treatment with Triton X-100, while the amount of M711 retained in the NPs remained almost unchanged (at ≈ 80% of total protein) (Fig. 4), in agreement with previous results (14). Taking this into account, a possible binding scheme could be the soft and hard corona model (42), in which proteins bound to the PHA NPs via the MinP tag are tightly bound to the NPs while those bound via unspeci c interactions of Cpl-711 are loosely attached.…”

“…For instance, a small peptide of 48 amino acids, MinP, was designed based on the binding moiety of phasin PhaF from Pseudomonas putida. MinP maintains the global binding capability of the whole protein (14). Phasin-mediated immobilization has been demonstrated both in vivo, where the PHA synthesis, protein production, and binding to the polymer take place simultaneously within the cytoplasm of the bacterial host; and in vitro, where proteins are immobilized onto previously formulated materials (14)(15)(16).…”

“…MinP maintains the global binding capability of the whole protein (14). Phasin-mediated immobilization has been demonstrated both in vivo, where the PHA synthesis, protein production, and binding to the polymer take place simultaneously within the cytoplasm of the bacterial host; and in vitro, where proteins are immobilized onto previously formulated materials (14)(15)(16). The in vitro approach is more suitable to ensure endotoxin removal from both the polymer and the protein when targeting biomedical applications (17), as well as to tightly control protein load.…”

Microbial Polyhydroxyalkanoates Granules: An Approach Targeting Biopolymer for Medical Applications and Developing Bone Scaffolds

“…Its versatility helps in the synthesis and depolymerizing of PHA molecules. Phasins are of high importance especially in biomedical applications because of their inherent ability of strong binding affinity to protein tags and also to PHA molecules, making them easily engineerable and helping in producing and performing biotechnological products for in vitro experiments [ 9 ]. Phasin protein is an important factor required to produce the nano sized PHA.…”

Los bioplásticos, ¿qué son? ¿cuántos hay? ¿cómo se producen?

“…Por tanto, la maquinaria metabólica involucrada en la producción del PHA implica una importante conexión de la sintasa y depolimerasa con otras rutas centrales y periféricas del metabolismo del carbono bacteriano, que son las que generan el monómero que va a formar parte del bioplástico. Gracias a la biología de sistemas, en la actualidad se están utilizando estrategias holísticas tanto para abordar su estudio como para desarrollar nuevos materiales con nuevas aplicaciones mediante ingeniería metabólica combinada con la biología sintética (4)(5)(6). En este sentido, merece la pena mencionar el bioplástico antibacteriano PHACOS que fue generado mediante ingeniería metabólica en la bacteria P. putida.…”