RT Journal Article
T1 Dissecting the polyhydroxyalkanoate-binding domain of the PhaF phasin: Rational design of a minimized affinity tag
A1 Mato, Aranzazu
A1 Blanco, Francisco
A1 Maestro García-Donas, María Beatriz
A1 Sanz, Jesús
A1 Pérez Gil, Jesús
A1 Prieto, María Auxiliadora
AB 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.
PB American Society for Microbiology
SN 0099-2240
YR 2020
FD 2020
LK https://hdl.handle.net/20.500.14352/95495
UL https://hdl.handle.net/20.500.14352/95495
LA eng
NO Mato ABlanco FGMaestro B, Sanz JMPérez-Gil J, Prieto MA 2020. Dissecting the Polyhydroxyalkanoate-Binding Domain of the PhaF Phasin: Rational Design of a Minimized Affinity Tag. Appl Environ Microbiol 86:e00570-20.https://doi.org/10.1128/AEM.00570-20
NO ACKNOWLEDGMENTSThis work was supported by the European Union’s Horizon 2020 Research and Innovation Program, grant agreement no. 633962 (P4SB), the Spanish Ministry of Science, Innovation and Universities (BIO2017-83448-R, BIO2016-79323-R, and RTI2018-094564-B-I00), and the Community of Madrid (P2013/MIT2807 and P2018/NMT4389). Francisco Blanco is the recipient of a predoctoral grant from the State Program for the Promotion of Talent and Its Employability in R&D&I (PRE-2018-083859) from the Spanish Ministry of Science and Innovation.This research was conducted by A. M. Aguirre in partial fulfillment of the requirements for a Ph.D. from the Universidad Complutense de Madrid, Madrid, Spain, 2019.
NO European Commission
NO Ministerio de Ciencia, Innovación y Universidades (España)
NO Comunidad de Madrid
DS Docta Complutense
RD 6 abr 2025