Leptospira interrogans thermolysin refolded at high pressure and alkaline pH displays proteolytic activity against complement C3

1. María Camila Molina Jiménez
Estudiante de medicina tercer semestre 2018-2

2. INTRODUCTION
• Leptospira interrogans
Spirochete
gram negative
Thermolysin
Proteolytic activity against
Complement C3 and C6
REFOLDING WITH
HHP AND
ALKALYNE PH

3. INTRODUCTION
• COMPLEMENT C3

4. OBJETIVE

5. MATERIALS AND METHODS
prothermolysin fragment PepSY-M4
Allows the transfer of DNA
fragments between different
cloning vectors while
maintaining the reading
frame.
transfer fragments of DNA between plasmids using a
patented set of recombination sequences, the
"Gateway att" sites and two patented enzyme
mixtures, called "LR Clonase" and "BP Clonase"
GATEWAY
Vectors and input clones
Donor Vectors
Recombination enzymes
Destination vectors
MultiSite Cloning
(LPF) Patoc
The maintenance of cells in vitro, preserving to
the maximum their physiological, biochemical
and genetic properties. Under the right
conditions
Culture
medium EMJH

6. MATERIALS AND METHODS
specific
detection of
molecules
the interaction of electromagnetic
radiation with matter. Through this
interaction the molecules can pass from
one energy state to another different
energy state.
SPECTROPHOTOMETER
emission process in which the molecules
are excited by the absorption of
electromagnetic radiation and when they
relax to the basal state they release the
excess energy in the form of photons.
quantitative
chemical
analysis
molecule
labeling

7. MATERIALS AND METHODS
Separate the dissolved substances in a
mixture by size, so that it produces
spots of different color in it.
contact two phases or mutually immiscible
components, which do not react chemically, one
of which is mobile and the other stationary.
graduated column or
chromatogram where each
chemical species has been
absorbed in a specific layer.
Evaluation of the proteolytic activity of thermolysin against C3
Separate the proteins according to their
electrophoretic mobility
migration of charged particles or solutes in a
liquid medium under the influence of an
electric field
polyacrylamide gel with sodium dodecyl
sulfate.
denatured proteins and distribution

8. MATERIALS AND METHODS
Detection of specific proteins
in a determinate sample
separation by size with electrophoresis
transfer to a solid support and finally visualization
through the labeling of proteins with the use of primary
or secondary antibodies

9. RESULTS

10. RESULTS

11. RESULTS

12. DISCUSSION
AUTHOR HYPOTHESIS
N.V MALAVASI
D. FOGUEL
2.4 kbar induces solubilization
of IBs and that incubation at 0.4
kbar is a condition in which
protein folding may occur but
reaggregation can be inhibited
T. ARAWAKA
D. EJIMA
K. TSUMUTO
M. UMETSU
There is similarity between the
effect of arginine and GdnHCl on
protein unfolding, although the
amino acid has lower denaturing
effect
A.K. Chang
J.W. Park
X. Gao, J. Wang
It has been described that
thermolysins present
autoproteolysis and that the
presence of the propeptides can
be inhibitors of their own
catalytic domain

13. CONCLUSIONS
The application of very little HHP and alkaline ph are not a good
method for efficient replication, however it avoided the powerful
denaturing, inactivation of proteins and reaggregation, so it can be
studied.
Proteolytic activity of the purified thermolysin generated a cleavage
profile of C3 in the LP, indicating the correct folding of the protein to a
biologically active form being a major target of therapeutic intervention