Page 100 - 00. Complete Version - Progress Report IPEN 2014-2016

100 Biotechnology | Progress Report

ary and tertiary structure of the insoluble and conversion of angiotensin I to the vasoconstric-
mostly inactive proteins produced in bacteria. tor angiotensin II and the hydrolysis of brady-
We demonstrated that high pressure can con- kinin (BK). Human somatic angiotensin-con-
vert insoluble aggregated proteins from IB to verting enzyme has two homologous domains
preparations with native tertiary structure (N and C) that share 60% identity, and the cat-
and fully biological activity with very high alytic site of the C-domain exhibits three-fold
yields (Fig. 4). Among the proteins that have greater activity than the N-domain in the hy-
been successfully refolded by our group are drolysis of angiotensin I in vivo. The catalyt-
the non-structural protein 1 (NS1) and enve- ic site of C-domain of angiotensin-converting
lope (protein E) proteins from dengue and zika enzyme peptide was expressed in a bacterial
viruses, endostatin, green fluorescent protein, system, and its purification was performed
a promising protein for Schistosoma mansoni in one step. Structural analysis by circular di-
vaccination (Sm29), the pentamer of subunit chroism and fluorescence confirmed that the
B of cholera toxin (CTB), among many others. purified protein is correctly folded, and cata-
lytic site of C-domain of angiotensin-convert-
Structural analyses of ing enzyme possesses enzymatic activity and

soluble human protein is inhibited by Lisinopril. This peptide can be
used to test new inhibitors and C-domain of
The L10 ribosomal protein (RPL10) plays a role angiotensin-converting enzyme substrates be-
in the binding of the 60 S and 40 S ribosom- cause this peptide is easy to produce and this
al subunits and in mRNA translation. The ev- has proven efficient link with these molecules
idence indicates that RPL10 also has multiple (“Structural characterization and enzymatic ac-
extra-ribosomal functions, including tumor tivity of the recombinant Ala959 to Ser1066
suppression and its overexpression osteoblasts region of human ACE”). Osteoblasts are spe-
exhibit a cell autonomous alteration that lead cialized fibroblasts that secrete and mineral-
to increased mineralization in vitro. We suc- ize the bone matrix, and there is little infor-
cessfully cloned and expressed full-length hu- mation on the fate and potential therapeutic
man RPL10 (hRPL10) protein and isolated in- efficacy of low-dose gamma-irradiation in the
clusion bodies that had been formed under formation of mineralization nodules in the os-
mild growth conditions. After the hRPL10 pu- teoblast culture. Our first results of low dose
rification using a two-step process of non-de- irradiated osteoblasts showed an increase in
naturing protein extraction from pelleted in- the number of mineralized nodules.
clusion bodies, we studied the characteristics
of this protein using circular dichroism spec-
troscopy and by monitoring the changes in-
duced by the presence or absence of zinc ions Biological effects of
using fluorescence spectrometry. The results ionizing radiation
suggested that the strategy used to obtain
hRPL10 is simple and could be applied to ob- Biological effects of ionizing radiation in aque-
taining other proteins that are susceptible to ous solution, produces several highly reactive
degradation (“A simple strategy for the puri- species. The most important are hydroxyl rad-
fication of native recombinant full-length hu- ical and hydrated electron. These products
man RPL10 protein from inclusion bodies”). interact with peptides and proteins causing
Angiotensin-converting enzyme catalyzes the several modifications such as fragmentation,

Instituto de Pesquisas Energéticas e Nucleares