2026-06-28T15:30:41Zhttps://docta.ucm.es/rest/oai/requestoai:docta.ucm.es:20.500.14352/954952025-03-18T14:07:32Zcom_20.500.14352_14col_20.500.14352_15
Docta Complutense
author
Mato, Aranzazu
author
Blanco, Francisco
author
Maestro García-Donas, María Beatriz
author
Sanz, Jesús
author
Pérez Gil, Jesús
author
Prieto, María Auxiliadora
2024-01-25T14:26:46Z
2024-01-25T14:26:46Z
2020
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
0099-2240
10.1128/AEM.00570-20
https://hdl.handle.net/20.500.14352/95495
1098-5336
https://doi.org/10.1128/AEM.00570-20
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.
eng
Attribution 4.0 International
Dissecting the polyhydroxyalkanoate-binding domain of the PhaF phasin: Rational design of a minimized affinity tag
journal article
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
URL
https://docta.ucm.es/bitstreams/82f51098-5407-408f-8022-e39920a6b672/download
File
MD5
d4bbd377101efcc8dd9be455b8359eae
820303
application/pdf
PhaF_Phasin-manuscript.pdf
URL
https://docta.ucm.es/bitstreams/849a51df-78f7-4a88-ae4e-50a0dceaa654/download
File
MD5
1bb09b8debf4c9d71fba83e78010b007
2329963
application/pdf
PhaF_Phasin-VoR.pdf
URL
https://docta.ucm.es/bitstreams/ab0a8538-38f3-4487-8684-218f14d3db54/download
File
MD5
a19e61f1b87e01a9b2ac2c7f67c1b3c2
71287
text/plain
PhaF_Phasin-manuscript.pdf.txt