Page 11 - PR 2014 2016 04 Biotechnology

Biotechnology | Progress Report 97

metic” qualifications. In recent years, interest aggregates in mild conditions, maintaining
in biomedical applications of natural and the existing native-like secondary and ter-
synthetic polymers has grown steadily, with tiary structure of the insoluble and mostly
a substantial contribution to the quality and inactive proteins produced in bacteria. We
duration of human life. Presently, novel po- demonstrated that high pressure can con-
rous biologically active composites based on vert insoluble aggregated proteins from IB to
hydroxyapatite (HA) and poly(caprolactone) preparations with native tertiary structure
(PCL) have been developed and tested, with and fully biological activity with very high
potential for use in scaffolds for bone tissue yields (Fig. 4). Among the proteins that have
engineering. The experiments are focused been successfully refolded by our group are
on the synthesis and biological response of the non-structural protein 1 (NS1) and enve-
bone to the PCL/HA composite. Such work lope (protein E) proteins from dengue and zika
resulted in a partnership with the Biosintesis viruses, endostatin, green fluorescent protein,
Company, which received a financial support a promising protein for Schistosoma mansoni
from FAPESP (PIPE project). vaccination (Sm29), the pentamer of subunit
B of cholera toxin (CTB), among many others.
Recombinant proteins –

Refolding from inclusion

bodies using high
hydrostatic pressure

A
Until the 1980s decade, the production of
proteins for therapeutic and research pur-
poses was obtained by purification from their
native sources. The production of proteins
was greatly facilitated by transgenic protein
expression, overcoming the difficulties of pu-
rification of proteins that were present at their
native sources usually very contaminated and
at low levels. The bacteria Escherichia coli is
the most efficient and cost-effective host for
recombinant heterologous protein produc- B
tion. However, E. coli is often unable to fully
process the recombinant foreign proteins
during overexpression and therefore mis-
folded proteins forms insoluble aggregated
proteins in bacterial cytoplasm, known as
inclusion bodies (IB). Solubilization of the IB
and the posterior refolding of the proteins
is necessary to produce active proteins from
IB. Utilization of high hydrostatic pressure
is a novel and robust method to disaggre- Figure 4. Scanning electron microscopy of cholera toxin
expressed in Escherichia coli inclusion bodies before (A)
gate proteins from IB, by solubilization of the and after (B) refolding with high pressure. Scale 5 μm.