Page 88 - Biodegradable Polyesters

66 3 Microbial Synthesis of Biodegradable Polyesters: Processes, Products, Applications

in vivo. The intein is a self-cleaving tag enabling release of the target protein after
PHA granule isolation. Grage et al. [75] fused the target protein to the PHA syn-
thase which is covalently linked to the PHA and hence more suitable as anchor
protein. The target protein was fused via an enterokinase cleavage site enabling
proteolytic release of the target protein from the PHA granules. HcRed (a flu-
orescent protein) and an anti-β-galactosidase scFv protein were purified using
this approach. PHA synthase as affinity tag is preferred as it avoids leakage of
uncleaved fusion protein.

3.8.7.4 Enzyme Immobilization
Immobilized enzymes are often more stable than the soluble counterparts and
can be recycled. They are also removable from a reaction mixture. PHA synthase
fusions with the enzyme of interest were successfully developed and enabled
production of recombinant enzyme already attached to a support material
(PHA) in one step. The β-galactosidase LacZ [21] was the first immobilized
enzyme followed by alpha-amylase, organophosphohydrolase, and enzymes
involved in sialic acid synthesis [76–78]. These demonstrated applicability PHA-
immobilized enzymes in food processing, biomass conversion, bioremediation,
and fine chemical synthesis.

3.8.7.5 Diagnostics and Imaging
The mouse interleukin-2 (IL2) or the myelin oligodendrocyte glycoprotein (MOG)
were anchored to PHA inclusions via fusion to phasins and subsequently sub-
jected to sequester antibodies in blood sera which binding was specifically and
sensitively detected using fluorescence-activated cell sorting (FACS) [66]. Detec-
tion of specific antibodies was possible up to a serum dilution of 1–100 000. In
another study, PHA inclusions co-displaying GFP and MOG fusion-generated flu-
orescent beads which were specifically binding anti-MOG and anti-GFP antibod-
ies [79]. Target protein displaying PHA beads were also attached to microtiter
plates for use in ELISA as was first demonstrated for binding IgG to protein A
domain displaying PHA beads [20].
Only recently, PHA inclusions were developed to display specific antigens of
pathogenic mycobacteria for use as skin test [80]. The PHA inclusions outper-
formed existing commercial offering in animal experiment and is currently be
tested in large-scale field trails.

3.8.7.6 Vaccine Delivery
Particulate subunit vaccines have become increasingly attractive as they target
antigen-presenting cells and enhance cellular immune responses. PHA inclusions
displaying selected antigens have been considered as vaccine candidates. PHB
beads displaying the HepC core antigen or mycobacterial antigens antigen-85A
(Ag85A) and 6 kDa early secretory antigenic target (ESAT6) based on fusions with
the PHA synthase were developed and produced [81–83]. In both cases, a strong
cell-mediated immune response was obtained which even led to protective immu-
nity as was shown by challenge with pathogenic mycobacteria.