Poly-3-Hydroxybutyrate Functionalization with BioF-Tagged Recombinant Proteins

Abstract

Polyhydroxyalkanoates (PHAs) are biodegradable polyesters that accumulate in the cytoplasm of certain bacteria. One promising biotechnological application utilizes these biopolymers as supports for protein immobilization. Here, the PHA-binding domain of the Pseudomonas putida KT2440 PhaF phasin (BioF polypeptide) was investigated as an affinity tag for the in vitro functionalization of poly-3-hydroxybutyrate (PHB) particles with recombinant proteins, namely, full-length PhaF and two fusion proteins tagged to BioF (BioF-C-LytA and BioF-β-galactosidase, containing the choline-binding module C-LytA and the β-galactosidase enzyme, respectively). The protein-biopolyester interaction was strong and stable at a wide range of pHs and temperatures, and the bound protein was highly protected from self-degradation, while the binding strength could be modulated by coating with amphiphilic compounds. Finally, BioF-β-galactosidase displayed very stable enzymatic activity after several continuous activity-plus-washing cycles when immobilized in a minibioreactor. Our results demonstrate the potentialities of PHA and the BioF tag for the construction of novel bioactive materials.IMPORTANCE Our results confirm the biotechnological potential of the BioF affinity tag as a versatile tool for functionalizing PHA supports with recombinant proteins, leading to novel bioactive materials. The wide substrate range of the BioF tag presumably enables protein immobilization in vitro of virtually all natural PHAs as well as blends, copolymers, or artificial chemically modified derivatives with novel physicochemical properties. Moreover, the strength of protein adsorption may be easily modulated by varying the coating of the support, providing new perspectives for the engineering of bioactive materials that require a tight control of protein loading.

Keywords: PHB; affinity tag; phasins; polyhydroxyalkanoates; protein immobilization.

FIG 1

(Left) Stability of PhaF binding to PHB at 25 and 37°C analyzed by SDS-PAGE. C, control of soluble protein; B, bound protein eluted from PHB with 1% (wt/vol) SDS after heating at 90°C for 10 min. (Right) Results of gel densitometry, shown as the percentages of the initial applied protein. Black solid bars indicate soluble control protein, whereas red hashed bars represent the particle-bound protein.

FIG 2

Elution of PhaF from different preparations of PHB upon incubation with detergents and the dependence on granule coating. (Left panel) SDS-PAGE analysis. C, control of soluble protein; SDS, 1% (wt/vol) SDS; Tr, 1% (vol/vol) Triton X-100; Tw, 1% (wt/vol) Tween 20; Ch, 3% (wt/vol) CHAPS; Co, 3% (wt/vol) sodium cholate; Sc, 3% (wt/vol) sarcosyl. (A) Naked PHB; (B) PHB coated with sodium oleate (PHBOL); (C) PHB coated with phosphatidylglycerol and phosphatidylcholine (PHBPL). (Right panel) Quantification of elution efficiency by gel densitometry.

FIG 3

Analysis of the saturation capacity of PHB preparations by Langmuir isotherm analysis. Each point in the plot represents the mean from three independent experiments.

FIG 4

SEM micrographs of PHB and PHBPL preparations.

FIG 5

(Left) Analysis by SDS-PAGE of the stability of BioF–C-LytA binding to PHB at 37°C. C, control of soluble protein; B, bound protein eluted from PHB with 1% (wt/vol) SDS after heating at 90°C for 10 min. Black arrows indicate degradation fragments of BioF–C-LytA in solution. (Right) Results of gel densitometry, shown as percentages of initial applied protein.

FIG 6

Activity of PHB-immobilized BioF–β-galactosidase during 16 continuous cycles. Time between cycles was 20 min, except for cycles 15 and 16, which were carried out after 48 and 96 h, respectively. Each experiment represents the mean from six determinations.