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

Aranzazu Mato,M Auxiliadora Prieto,Francisco G Blanco,Jesús Pérez-Gil,Jesús M Sanz,Beatriz Maestro,Maia Kivisaar

doi:10.1128/aem.00570-20

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 coliIMPORTANCE 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.

Highlights

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
Many granuleassociated proteins (GAPs) have been exploited as tags for the functionalization of PHA, especially PHA synthases and phasins
Despite only a few studies having addressed the structural aspects of phasins, it is known that some common characteristics include the high proportion of disordered regions [28, 37], the presence of residues in ␣-helical conformation that increases in the presence of PHA [9, 28], and their tendency to form oligomers in solution [8, 28, 29, 38]

Summary

Introduction

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. Among GAPs, phasins constitute the most abundant group of proteins covering the granule [8, 9] and have been widely used as anchoring tags to functionalize PHA supports for a variety of applications, such as affinity bioseparation [10], protein purification [11,12,13], enzyme immobilization [14,15,16], protein delivery to natural environments [17], diagnostics [18, 19], cell targeting [20,21,22,23,24], and antimicrobials [25]

Methods

Results

Conclusion