Tolerance of theRalstonia eutrophaClass I Polyhydroxyalkanoate Synthase for Translational Fusions to Its C Terminus Reveals a New Mode of Functional Display

Abstract

Here, the class I polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha was investigated regarding the functionality of its conserved C-terminal region and its ability to tolerate translational fusions to its C terminus. MalE, the maltose binding protein, and green fluorescent protein (GFP) were considered reporter proteins to be translationally fused to the C terminus. Interestingly, PhaC remained active only when a linker was inserted between PhaC and MalE, whereas MalE was not functional. However, the extension of the PhaC N terminus by 458 amino acid residues was required to achieve a functionality of MalE. These data suggested a positive interaction of the extended N terminus with the C terminus. To assess whether a linker and/or N-terminal extension is generally required for a functional C-terminal fusion, GFP was fused to the C terminus of PhaC. Both fusion partners were active without the requirement of a linker and/or N-terminal extension. A further reporter protein, the immunoglobulin G binding ZZ domain of protein A, was translationally fused to the N terminus of the fusion protein PhaC-GFP and resulted in a tripartite fusion protein mediating the production of polyester granules displaying two functional protein domains.

Vaccines Displaying Mycobacterial Proteins on Biopolyester Beads Stimulate Cellular Immunity and Induce Protection against Tuberculosis

“…It is also likely that increased antigen could be displayed on beads by incorporating multiple gene repeats in the bacterial production strains (31). Another strategy which utilized both N-terminus and C-terminus fusions of PhaC (19) could be applied to enable production of two different vaccine antigens on the one bead and therefore produce multivalent vaccines. Combinations of PHB bead vaccines and BCG could result in the induction of enhanced protection against TB.…”

New Skin Test for Detection of Bovine Tuberculosis on the Basis of Antigen-Displaying Polyester Inclusions Produced by Recombinant Escherichia coli

“…Proteins attached to the polyester beads were separated by SDS-PAGE using 8% polyacrylamide gels and stained with Coomassie blue. Proteins of interest were excised from the gels and subjected to tryptic peptide fingerprinting using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) (17).…”

“…Examples of foreign protein functions which have been successfully displayed on polyester beads using PhaC engineering are enzymes, binding domains/peptides, and antigens (13)(14)(15). Simultaneous fusions of several foreign proteins as well as coproduction of different PhaC fusion proteins enabled the codisplay of different protein functions on the same polyester bead with defined stoichiometry of the various proteins (12,(16)(17)(18). Foreign target proteins have been displayed on polyester beads for subsequent cleavage and purification (19)(20)(21)(22).…”

Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones

“…It has been reported that modifications to the C terminus of PhaC Re can be introduced without affecting enzyme activity only if the hydrophobic environment surrounding the C terminus is maintained (20). To test whether the coexpression of chaperones could prevent the reduction in PhaC Re activity caused by a C-terminal His 6 tag, the experiment was repeated using pASG1phaC Re to express a C-terminally tagged protein.…”