Seminario del artículo "Production of soluble bioactive mouse leukemia inhibitory factor from Escherichia coli using MBP tag"

1. Laura Durango Villada
Estudiante Medicina
Tercer semestre 2018-02

2. INTRODUCTION
E. Coli
◦ Gram-negative bacterium
with bacillus form
◦ It’s found in lower intestine
of warm blooded
organisms
◦ It’s very important for
digestion process
◦ Some strains can be
infectious when exchange
their genetic material
MBP: Maltose-binding protein
◦ Protein in E.Coli, encoded by the
malE gene, which is responsible for
the catabolism of maltodextrin.
◦ Enhance the solubility of a variety
of target proteins
◦ Stabilize and protect its
downstream passenger protein
from proteolytic degradation
during and after protein synthesis .

3. INTRODUCTION
Leukemia
◦ It’s a cancer of the early
blood-forming cells. Result
in high number of
abnormal white blood cells
◦ The type of leukemia
depends on the type of
the blood cells that
becomes cancer

4. OBJETIVE:
Produce a soluble
bioactive mouse leukemia
inhibitory factor from
Escherichia Coli using MBP
tag.

5. METHODS AND MATERIALS
Plasmid:
◦ Plasmids are small extra chromosomal
elements that can be transferred
between bacteria and can be
replicated in a host cell

6. PCR
Technique for
amplifying a gene, a
fragment of DNA by
means of an in vitro
enzymatic reaction
increases a specific
segment of DNA using
a polymerase, which
has specific activity of
synthesis, repair and
ability to extend or
shorten DNA, to obtain
millions of copies
What for? Fundament
RT-PCR
PCR variant. Uses RNA
as a template, allows
the detection and
amplification of RNA
The RNA is reverse
transcribed into
complementary DNA
(cDNA) using a reverse
transcriptase.

7. SDS
PAGE
MTT
It’s technique that
involves electrophoresis
and gels, which can
vary in composition
and pore size
What for?
Is used to separate proteins
by electrophoresis, which is
the migration of charged
particles or solutes in a liquid
medium, under the
influence of an electric field
It’s a assay for assessing
cell metabolic activity
This assay uses NADPH-dependent
cellular oxidoreductase enzymes
to reflect the number of viable
cells present. These enzymes are
capable of reducing the MTT to its
insoluble formazan, which has a
purple color

8. RESULTS
PCR amplification of mLIF gene was
performed routinely and 560 bp bands
in electro- phoresis that shows the
correct amplification of mLIF gene
Fig. 1

9. Analyze expression and purification of
MBP-mLIF using 10% SDS-PAGE
Fig. 2

10. Biological function assay of MBP-
mLIF on maintaining the
pluripotency iPSC cells
Fig. 3

11. DISCUSSION

12. CONCLUSION
The method proposed by the
authors to create a bioactive
mouse LIF recombinant
proteins in E.coli open the
doors to more researchs
because the costs decreased
when proposing jus one-step
purification of MBP-mLIF
Although this method is easier
and promising, the need to
develop new techniques that
avoid an immune response in
vivo is obvious