Running title: Rational design of a minimized PHA affinity tag

52 p.-9 fig.-3 tab.Phasin PhaF from Pseudomonas putida consists of a modular protein whose N-terminal domain (BioF) has been demonstrated to be responsible for the binding to the PHA granule. BioF has been exploited with 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 have analysed the mean hydrophobicity and the hydrophobic moment of the native BioF tag to rationally design shorter versions that maintain the affinity for the granule. Hybrid proteins containing the green fluorescent protein (GFP) fused to the BioF derivatives were studied for in vivo localization on the PHA, stability on the surface of the PHA granule against pH, temperature and ionic strength, and their possible influence on the PHA synthesis. Based on the results obtained, a minimized BioF tag for PHA functionalization has been proposed (MinP), which retains similar binding properties but possesses an attractive biotechnological potential derived from its reduced size. The MinP tag was further validated by analyzing functionality and stability of the fusion proteins MinP-β-galactosidase and MinP-CueO from Escherichia coli.IMPORTANCE.Polyhydroxyalkanoates (PHAs) are biocompatible, non-toxic 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.This work was supported by the European Union’s Horizon 2020 Research and Innovation Programme, grant agreement no. 633962 (P4SB), the Spanish Ministry of Science, Innovation and Universities (BIO2017-83448-R, BIO2016-79323-R, RTI2018-094564-B-I00) and the Community of Madrid (P2013/MIT2807, P2018/NMT4389). Francisco Blanco is a recipient of a predoctoral grant from the State 560 Program for the Promotion of Talent and its Employability in R&D&I (PRE-2018- 561 083859) from the Spanish Ministry of Science and Innovation.Peer reviewe