In vivo immobilization of fusion proteins on bioplastics by the novel tag BioF

Abstract

A new protein immobilization and purification system has been developed based on the use of polyhydroxyalkanoates (PHAs, or bioplastics), which are biodegradable polymers accumulated as reserve granules in the cytoplasm of certain bacteria. The N-terminal domain of the PhaF phasin (a PHA-granule-associated protein) from Pseudomonas putida GPo1 was used as a polypeptide tag (BioF) to anchor fusion proteins to PHAs. This tag provides a novel way to immobilize proteins in vivo by using bioplastics as supports. The granules carrying the BioF fusion proteins can be isolated by a simple centrifugation step and used directly for some applications. Moreover, when required, a practically pure preparation of the soluble BioF fusion protein can be obtained by a mild detergent treatment of the granule. The efficiency of this system has been demonstrated by constructing two BioF fusion products, including a functional BioF-beta-galactosidase. This is the first example of an active bioplastic consisting of a biodegradable matrix carrying an active enzyme.

FIG. 1.

Schematic final protocol for the immobilization (PFprotein) and purification (Fprotein) of BioF products.

FIG. 2.

Analysis of GAPs from P. putida GPo1. The image shows an SDS-PAGE (12.5%) analysis of GAPs isolated by different methods. Lane 3, ultracentrifugation through a sucrose gradient; lane 4, centrifugation (12,000 × g, 4°C) onto a discontinuous glycerol gradient; lane 5, simple centrifugation (4,000 × g, 30 min); lane 2, total crude extract of P. putida GPo1; lane 1, standard molecular size markers.

FIG. 3.

Outline of shuttle plasmid pNFA2 and immobilization and purification of FLyt product. (A) Construction of a shuttle plasmid expressing the flyt gene. Hatched arrows represent the DNA region coding for the ChBD. Black boxes represent the DNA region coding for the BioF tag. Abbreviations: Ev, EcoRV; H, HindIII; HII, HindII; Xb, XbaI; Plac, lac promoter of pUC18; Ptac, tac promoter of pVLT35. (B and C) SDS-PAGE (12.5%) analysis of FLyt production, immobilization, and purification process. Lane 1, soluble fraction of crude extract of P. putida GPG-Tc6; lane 2, complete (soluble and insoluble) crude extract of P. putida GPG-Tc6; lane 3, soluble fraction of crude extract of P. putida GPG-Tc6(pNFA2); lane 4, complete (soluble and insoluble) crude extract of P. putida GPG-Tc6(pNFA2); lane 5, GAPs of P. putida GPG-Tc6(pNFA2), with the FLyt protein immobilized; lanes 6 to 9, soluble granule fractions after treatment with detergents (lane 6, 0.15% Tween 20; lane 7, 0.15% sodium deoxycholate; lane 8, 0.15% Sarkosyl; lane 9, 0.15% Triton X-100). The molecular masses of the standard marker proteins are indicated. The position of the FLyt protein is indicated with arrows.

FIG. 4.

Construction of shuttle plasmid pNFL2 and immobilization and purification of the enzyme FLac. (A) Construction of a shuttle plasmid that codes for the flac gene. White arrows represent the DNA region coding for the reporter lacZ. Black boxes represent the DNA region coding for the BioF tag. Abbreviations: Ev, EcoRV; H, HindIII; Sm, SmaI; Xb, XbaI; Plac, lac promoter of pUC18; Ptac, tac promoter of pVLT35. (B and C) SDS-PAGE (10%) analysis of FLac production, immobilization, and purification process. Lane 1, soluble fraction of crude extract of P. putida GPG-Tc6(pNFL2); lane 2, complete (soluble and insoluble) crude extract of P. putida GPG-Tc6(pNFL2); lane 3, GAPs of P. putida GPG-Tc6(pNFL2), with the FLac protein immobilized; lane 4, insoluble granule fraction after treatment with 0.15% Sarkosyl; lane 5, soluble granule fraction after treatment with 0.15% Sarkosyl. The position of the FLac protein is indicated with arrows. The molecular masses of the standard marker proteins are indicated.

FIG. 5.

Multiple sequence alignment of PhaF-like proteins. A comparison of the amino acid sequences of the N-terminal domain of the PhaF protein of P. putida GPo1 (CAA09109) and those of other proteins in the databases that presented significant similarity, including the homologous proteins to PhaI, is shown. The numbers at the right indicate the positions of the residues in the complete amino acid sequence of the protein. A consensus sequence was deduced for positions at which the residues were identical in at least 7 of the 14 compared sequences. Black shading indicates the hydrophobic region that is conserved in all PhaF-like proteins. The accession numbers correspond to PhaF-like proteins from the following microorganisms: AAG08445, Pseudomonas aeruginosa PA01; BAB91367, Pseudomonas sp. strain 61-3; BAB78723, Pseudomonas chlororaphis; ZP_00084249, Pseudomonas fluorescens; CAA09109, P. putida GPo1; AAG08446, P. aeruginosa PA01; BAB91366, Pseudomonas sp. strain 61-3; BAB78724, P. chlororaphis; ZP_00084248, P. fluorescens; CAA09108, P. putida GPo1; NP_298564, Xylella fastidiosa 9A5C; ZP_00040599, X. fastidiosa ANN-1; NP_636955, Xanthomonas campestris pv. campestris ATCC 33913; NP_641975, X. campestris pv. citri 306.