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Dissecting the Polyhydroxyalkanoate-binding domain of the PhaF Phasin: rational design of a minimized affinity tag Mato, Aranzazu Blanco, Francisco G. Maestro García-Donas, María Beatriz Sanz, Jesús M. Pérez-Gil, Jesús Prieto, M. Auxiliadora 577.112 57.08 Biotechnology Functionalization Phasins Polyhydroxyalkanoates Pseudomonas putida Biotecnología Bioquímica (Biología) 3399 Otras Especialidades Tecnológicas 2302 Bioquímica 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 -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–-galactosidase and MinP-CueO from Escherichia coli. Unión Europea. Horizonte 2020 Ministerio de Ciencia e Innovación (MICINN) Comunidad de Madrid Sección Deptal. de Bioquímica y Biología Molecular (Biológicas) Fac. de Ciencias Biológicas TRUE pub 2023-06-16T15:19:03Z 2023-06-16T15:19:03Z 2020-06-02 journal article https://hdl.handle.net/20.500.14352/6323 0099-2240, ESSN: 1098-5336 10.1128/AEM.00570-20 eng P4SB (633962) (BIO2017-83448-R, BIO2016-79323-R, and RTI2018-094564-B-I00) (PRE-2018-083859) (P2013/MIT2807 and P2018/NMT4389) Atribución 3.0 España https://creativecommons.org/licenses/by/3.0/es/ open access application/pdf American Society for Microbiology