Phuong Pham
Dr. Gary Merrill
Summer 2011
 Explore
the functions of thioredoxin
reductase
 Only
known enzyme to reduce thioredoxin
 Recent
research suggested roles in
reducing toxic carbonyl-containing
compounds in cells
http://www.molecularstructure.org/entry.php?pdb=1ZKQ

Small protein
(104 amino acids)

Supplies electrons for
enzymatic and regulatory
reactions

Reduced again by
thioredoxin reductase
http://en.engormix.com/MA-dairy-cattle/articles/selenium-metabolism-animals-relationship-t363/p0.htm

Recent studies suggests it
also reduces carbonyls
(ketones and aldehydes)

Two active sites
(site 1 near N terminus; site 2
near C terminus)

Site 2 has the unusual
amino acid selenocysteine
http://www.asiaandro.com/archive/1008-682X/5/231.htm

Mechanism of
thioredoxin reduction:
NADPH FAD  Site 1
Site 2 Trx

Mechanism of carbonyl
reduction is unknown
http://www.asiaandro.com/archive/1008-682X/5/231.htm
 Only
site 1 of thioredoxin reductase is
needed for carbonyl reduction
 The
selenocysteine-containing site 2 is
unnecessary

In eukaryotes, the amino acid selenocysteine
(Sec) is incorporated opposite UGA codon

Because bacteria cannot insert Sec opposite
UGA, site 2 is inactive

Recombinant Txnrd1 cannot reduce thioredoxin
http://www.edvotek.com/300

To obtain active recombinant Txnrd1 protein, the Sec codon is
changed to a cysteine (Cys) codon

The Cys form of Txnrd1 is 10 times less active than wild type form
in reducing thioredoxin

However, this form is fully active in reducing the carbonylcontaining compound menadione
 Site 2 might not be necessary in
reducing carbonyls
Wild Type Txnrd1 sequence expressed in eukaryotic cells
Met
X
X……… X
Gly
Cys
Sec
Gly
Reduces thioredoxin and carbonyls
Wild Type Txnrd1 sequence expressed in E. coli
Met
X
X……… X
Gly
Cys
Lower level of thiorexin reduction; still reduces carbonyls
Engineered Txnrd1 sequence expressed in E. coli
Met
X
X……… X
Gly
Cys
STOP
Cys
Gly

Reduces Glutathione

Similar dimer structure
to Txnrd1

Participates in DNA
synthesis

Defends against
oxidative stress
http://www.asiaandro.com/archive/1008-682X/5/231.htm

Compare TR carbonyl reduction of mammals against yeast & bacteria

Compare TR carbonyl reduction ability against GR

Carbonyl reduction is specific to TR in mammals?
Reduction of
Mouse TR Yeast TR
Yeast GR
E. coli TR
E. coli GR
Trx


N/A

N/A
Grx
N/A
N/A

N/A


?
?
?
?
Carbonyl

Generated mTR1 insert through PCR reaction

Inserted into TOPO cloning vector and transformed with E. coli

Performed PCR to determine orientation of mTR1 insert

Cut insert out with Nde1 and BamH1 restriction enzymes

Inserted into pET28a expression vector and transformed with E. coli

Digested with restriction enzymes to verified presence of vector
and insert and transformed into BL21 Competent E. coli

Harvested enzyme using TALON Metal Affinity Resins

Added Nde1 restriction
site at beginning of the
3000
insert
2000
~1500 bp

Verified presence of insert
on electrophoresis gel
1000

Approx. 1500 base pairs

Generated mTR1 insert through PCR reaction

Inserted into TOPO cloning vector and transformed with E. coli

Performed PCR to determine orientation of mTR1 insert

Cut insert out with Nde1 and BamH1 restriction enzymes

Inserted into pET28a expression vector and transformed with E. coli

Digested with restriction enzymes to verified presence of vector
and insert and transformed into BL21 Competent E. coli

Harvested enzyme using TALON Metal Affinity Resins
http://www.nmr.chem.uu.nl/users/rob/efc.html

Before expressing to
A
obtain more genes

Taq polymerase adds
single A to insert’s 3’
ends

Transformed with
Top10 Competent
E. coli
http://www.clas.ufl.edu/jur/200209/papers/paper_henry.html
mTR1 insert
A

Generated mTR1 insert through PCR reaction

Inserted into TOPO cloning vector and transformed with E. coli

Performed PCR to determine orientation of mTR1 insert

Cut insert out with Nde1 and BamH1 restriction enzymes

Inserted into pET28a expression vector and transformed with E. coli

Digested with restriction enzymes to verified presence of vector
and insert and transformed into BL21 Competent E. coli

Harvested enzyme using TALON Metal Affinity Resins
pTOPO-mTR1 
pLac
m13r

AUGf 
mTR1 insert
~1500bp
m13f

 TGAr
Nde1
TOPO Vector
3.9 kb
pTOPO-mTR1 
pLac
m13r

AUGf
mTR1 insert
~1500bp
TGAr 
TOPO Vector
3.9 kb
Nde1
m13f

Primers
Predicted PCR
Product

TGAr + m13r
1500 bp

TGAr + m13f
No product

TGAr + m13r
No product

TGAr + m13f
1500 bp
Orientation

Insert is in the
1
reverse orientation in
7 8
TGAr + m13f
Chose the 7th and 9th
1
2
3 4
~1500 bp
5 6
7 8
9
Note: Abnormality in
all clones have same
orientation
9
1000
clones

6
2000
plasmid

2 3 4 5
2000
No Product
1000
TGAr + m13r

Generated mTR1 insert through PCR reaction

Inserted into TOPO cloning vector and transformed with E. coli

Performed PCR to determine orientation of mTR1 insert

Cut insert out with Nde1 and BamH1 restriction enzymes

Inserted into pET28a expression vector and transformed with E. coli

Digested with restriction enzymes to verified presence of vector
and insert and transformed into BL21 Competent E. coli

Harvested enzyme using TALON Metal Affinity Resins
pLac
m13r

BamH1
253
1496
AUGf
mTR1 insert
~1500bp
TGAr 
Nde1
m13f

EcoR5
314
TOPO Vector
3.9 kb
Used Nde1 and BamH1 restriction enzymes
to cut out insert

Generated mTR1 insert through PCR reaction

Inserted into TOPO cloning vector and transformed with E. coli

Performed PCR to determine orientation of mTR1 insert

Cut insert out with Nde1 and BamH1 restriction enzymes

Inserted into pET28a expression vector and transformed with E. coli

Digested with restriction enzymes to verified presence of vector
and insert and transformed into BL21 Competent E. coli

Harvested enzyme using TALON Metal Affinity Resins

Readied insert for
expression

Used pET28a already
cut at Nde1 and BamH1
sites

Transformed with DH5α
competent E. coli
http://www.genomex.com/vector_maps/pET28_map.pdf
pET28a
5.4 kb

Generated mTR1 insert through PCR reaction

Inserted into TOPO cloning vector and transformed with E. coli

Performed PCR to determine orientation of mTR1 insert

Cut insert out with Nde1 and BamH1 restriction enzymes

Inserted into pET28a expression vector and transformed with E. coli

Digested with restriction enzymes to verified presence of vector
and insert and transformed into BL21 Competent E. coli

Harvested enzyme using TALON Metal Affinity Resins
1
2
3
4
5
6
7
8

Clones 7 and 16
weakly showed
~1500 bp insert
2000
1000

9
Proceeded with
transformation into
10 11 12 13 14 15 16
BL21 E. coli
2000
1000

Also used clone 5 as
control
Uninduced
5
7
IPTG Induced
16
5
7
16
kD
250
150
100
53 kD for
75
Txnrd1
50
according
37
to
25
literature
20
15
10

Generated mTR1 insert through PCR reaction

Inserted into TOPO cloning vector and transformed with E. coli

Performed PCR to determine orientation of mTR1 insert

Cut insert out with Nde1 and BamH1 restriction enzymes

Inserted into pET28a expression vector and transformed with E. coli

Digested with restriction enzymes to verified presence of vector
and insert and transformed into BL21 Competent E. coli

Harvested enzyme using TALON Metal Affinity Resins

Used the TALON resin
beads with cobalt to
bind to polyhistidine
tag on proteins

Unsuccessful in binding
protein to resin
Conclusion: Proteins might be insoluble
or not able to bind to resins
http://www.clontech.com/US/Support/Applications/His-Tagged_Protein_Purification/Ni-NTA_Resin_vs._Talon?sitex=10020:22372:US
Wild Type Txnrd1 sequence expressed in eukaryotic cells
Met
X
X……… X
Gly
Cys
Sec
Gly
Re-engineered Txnrd1 sequence expressed in E. coli
Met
X
X……… X
Gly
Replace Cys and Sec with Serine (Ser)
Ser
Ser
Gly

Continue to express newly truncated gene and purify
the protein

Can observe cellular activity through microscopy of
fluorescent staining and morphology

Identify the mechanism of carbonyl reduction in
thioredoxin reductase

Dr. Gary Merrill

Dr. Kevin Ahern

Francis Cripps Foundation

Environmental Health
Sciences Center

Howard Hughes Medical
Institute