Transcript Production of empty Turnip yellow mosaic virus capsids from

Production of Turnip yellow
mosaic virus nano-containers
from Lactococcus lactis for zinc
fortification
Alma Laney
Dr. Theo Dreher Lab
Department of
Microbiology
Nutrition in America
• Many Americans, 10%, have a deficiency
in zinc levels, somewhat due to the
negative interactions with grains and
beans which causes the zinc to become
unavailable for use.
• Zinc is part of the active site for many
enzymes.
• Zinc is especially essential for pregnant
and nursing mothers.
TYMV capsids for micronutrient
delivery
• Lactococcus can be modified to grow
cheese and yogurt that is zinc enriched
and help solve the problem of zinc
deficiency.
• The problem is how to protect the zinc
from chelation by phytates during
digestion.
• What if the zinc was protected by a shell
that would eventually break down later in
digestion process?
Turnip yellow mosaic virus could
solve this problem
• The outer and inner surface of TYMV
capsids contain several sites for
modification by chemical reactions.
Turnip yellow mosaic virus
general background
• The TYMV capsid
is made of 180
copies of the
same protein
monomer.
• The capsid
icosahedral and
28 nm in diameter
• Infects crucifers
What is a minimal zinc finger?
• A minimal zinc
finger is the
minimal number
(25) of amino
acids required
to effectively
bind zinc and be
functional.
Project overview
• Production and purification of TYMV
capsids in E. coli
• Production in Lactococcus
• Creating a minimal zinc finger within the
TYMV capsid.
• Future work
Predictions
• The TYMV empty capsids can be formed
in E. coli and be purified.
• The TYMV empty capsids can also be
produced by Lactococcus.
• The zinc finger will not interfere with
capsid assembly.
Production and purification in E.
coli
• The monomer has
been expressed in E.
coli BL21 previously.
• The empty capsid was
also produced and
purified from E. coli
pLysS.
Production in E. coli
Nde I [Start of transcription]
Stop of transcription [100
bp] upstream from Hind III
TYMV CP [570bp]
Hind III
Production cont.
• The capsid was induced using 1mM IPTG
at 28 degrees C for 9 or more hours.
IPTG
RNA
Pol
Repressor
Lac promoter
TYMC gene
The cells were then lysed using lysozyme and
sonication.
The capsids were then purified by size exclusion
using a Centricon.
Purification in E. coli
Lane 1: Protein ladder
Lane 2: Pre-induced
Lane 3: Post-induced
Lane 4: Insoluble fraction
Lane 5: Soluble fraction
Lane 1 2
120 kDa
86 kDa
47 kDa
34 kDa
26 kDa
21 kDa
20 kDa
3
4
5
Production in Lactococcus
• The TYMC gene was amplified using
PCR.
• The PCR primers contained restriction
sites to expedite ligation into the shuttle
vector.
Pst I
TYMV CP
Xba I
The shuttle vector
• The shuttle vector is pBG568 pipwt.
Pst I
OriC
Xho I
Pip wt gene
Xba I
Ori [Lactococcus]
Erythromycin
Continuing work in Lactococcus
• The TYMV capsid gene will then be ligated
into pBG568, a shuttle vector that contains
the ori for both E. coli and Lactococcus.
• The sequence will then be verified by
restriction digest and sequencing the gene
of interest.
• The Lactococcus will then be induced to
produce the empty TYMV capsid.
Continuing work in Lactococcus
• The capsid will then be purified and
verified as correct by electron microscopy
and by SDS-PAGE gel electrophoresis.
Continuing work in E. coli
• The capsid gene will then be altered with a
minimal zinc finger inside the formed
capsid.
Zinc finger addition
• The zinc finger was generated using megaprimers that include the start of the TYMV CP
• The product then had 20 cycles of PCR to
amplify the sequence and to place restriction
sites for insertion into the TYMV CP.
Minimal zinc finger sequence
+ start of TYMV CP
Pvu II
Nde I
Nde I
Pvu II
TYMV CP
Zinc finger cont.
Zinc
finger
insert
[~190bp]
Continuing work
• The empty zinc modified capsid will then
be purified and checked for zinc content.
Future work
• Produce empty
capsids of different
sizes.
• Produce cheese that
has been zinc
enriched.
• Test to see if the
TYMV-zinc capsids
will survive the
stomach and be
digestible in the small
intestine.
Acknowledgements
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The Howard Hughes Medical Institute
Dr. Theo Dreher
Dr. Bruce Geller
Josh Powell
Dr. Yannis Tzanetakis
The Theo Dreher Lab