1. Methodology: Plasmid preparation
• Primers for the double stop codon TGATAA, omega GFP, Y13 and Y49 mutants, for pSup35(1-40) and (1-80)
were designed, based on the genetic code of Escherichia coli, with the addition of GFP, Sup35 (yeast),
Strep 2 and 6xHistidine tag.
• Plasmids were mutated through site-directed mutagenesis (using QuickChange kit protocol).
• Plasmid lengths checked via 1% agarose gel.
• Competent Escherichia coli transformed with the obtained plasmid. Transformed cells incubated overnight at
37oC.
• Two colonies inoculated into 3ml LB broth (+1X Ampicillin). These were incubated the following night.
Plasmids purified and mutations confirmed via DNA sequencing.
• Subcloning with the original plasmid was done to make sure no unwanted mutations took place.
• Restriction digestion, followed by DNA sequencing confirmed our mutations and the integrity of the rest of the
plasmid.
• From there, we repeat mutagenesis, this time for GFPΩ, onto TGATAA-mutated plasmid (40 only, as 80
mutagenesis failed). Result was confirmed in a similar manner (plasmid purification, digestion, sequencing).
Mutagenesis for GFPΩ on TGATAA was done because later on, we could not cut out GFPΩ, without the
double stop codon.
• Mutagenesis was repeated for Y13 in (1-40), Y13 and Y49 in (1-80).
• As GFPΩ(80) failed, we used subcloning to insert GFPΩ(40) into Sup35(80)
• We combine TGATAA with Y13(40), Y13(40) and Y(49) through subcloning.
• Competent cells were transformed with the mutations and results were confirmed with restriction-digestion
and sequencing.
- Each of the plasmids (Ampicillin resistant) were transformed into OverExpress C41(DE3) competent
cells containing pSupBpaRS-6TRN (Chloramphenicol/Cam resistant). Cells were then plated on LB
plates supplemented with Cam and Amp, and incubated at 37°C overnight.
Overnight colonies carried a tRNABpaCUA and amino-acyl tRNA synthetase specific to pBpa
(BpaRS). The BpaRS will aminoacylate the tRNABpaCUA, the anticodon of which is complementary
to amber condon.
Protein expression
Three colonies from each plate were inoculated into LB/Amp/Cam/pBpa broth, LB/Amp/pBpa (-Cam)
broth and LB/Amp/Cam (-pBpa) broth respectively. The latter two acted as controls.
After incubation at 37°C, 120 µl of overnight culture were subcultured, until OD600=0.6-0.8. A final
concentration of 1mM IPTG was added to the media, to induce expression of BpaRS/tRNA pair and
Sup35-GFP-StrepII-His mutants. Induction and incubation (37oC) took place for 3h. The cell-lysate
proteins were visualized by InstantBlue Dye via SDS-PAGE.
Results and discussion:
• Post induction, GFP mutants were viewed under the microscope. Following images were taken,
showing that GFP functionality (Figure4 & 5. a) – CAM, b) – pBpa, c) Fully induced):
• SDS-PAGE results of mutated cells show expected bands, plus background proteins. For comparison
to the original induced Sup35, refer to Figure 5
aReference:
1. Addition of a photocrosslinking amino acid to the genetic code of Echerichia coli, J. W. Chin, A. B.
Martin, D. S. King, L. Wang, P. G. Schultz; 2002, Proceedings of the National Academy of
Sciences of the United States of America.
2. Benzophenone Photophores in Biochemistry, G. Dormán, G.D. Prestwich; 1994, Biochemistry
3. Prion, Wikipedia, https://en.wikipedia.org/wiki/Prion
4. The site-specific incorporation of p-iodo-L-phenylalanine into proteins for structure determination, J.
Xie, L. Wang, N. Wu, A. Brock, G. Spraggon, P. G. Schultz; 2004, Nature Biotechnology
In vivo incorporation of para-benzoyl-l-phenylalanine,
an artificial amino acid, into protein Sup35, a yeast prion
Tomasz Pawlak, Chan Wai Shan and Chih-Yen King
1. Tyrosine codons
TAC, to be mutated
Figure 2: Original inserts, contain yeast Sup35 fragments, GFP, Strep II (purification), His tag
(purification), Amber/Ochre - original stop codons
Introduction:
Prions are proteins with multiple structurally unique
conformations, at least one of which can recruit
other, correctly folded prion proteins, resulting in the
formation of protein aggregates. These misfolded
proteins form amyloid fibrils. Formation and
propagation of these fibrils is closely associated with
several pathologies, such as Alzheimer’s Disease,
Kuru, Huntington’s Disease and Parkinson’s Disease
in humans and “Mad Cow’s” Disease (Bovine
Spongiform Encephalopathy) and Scrapie in other
mammals – cows and sheep respectively. These
protein-like infectious particles have fungal
equivalents, which can be used to investigate and
model folding patterns and progression of amyloids in
general. Sup35 is a translation termination factor in
S. cerevisiae which forms a complex with other
release factors.
Sup35 recognizes a stop codon, initiating polypeptide
release.
When infected with [PSI+], a self propagating
misfolded variant of Sup35 (201 amino acids), the
volume of functional Sup35p decreases, potentially
allowing translation to continue. This can result in a
non-functional, nonsense protein. Depending on the
genetic background, [PSI+] yeast can fare better
than non-infected yeast. [PSI+] yeast, still capable of
reproducing, infects its daughter cells. In a sharp
contrast to mammalian prion diseases, yeast prions
can be a positive influence on the cell. In this project,
Sup35 fragments (1-40) and (1-80), which are
capable of infecting “healthy” proteins were mutated,
as to allow an unusual amino acid, pBnp (para-
benzoyl-l-phenylalanine) to be incorporated into the
segments
Incorporation of unnatural amino acids, such as
photo linkers or heavy ions is a useful tool in
investigating protein interactions. Para-Bpa is capable
of cross-linking one Sup35 fragment to another,
proximal and sterically accessible peptide backbone,
under UV and was chosen due to the high fidelity of
that reaction. In (1-40) and (1-80), Tyr13 and Tyr13 &
49 respectfully, were changed to amber codons (UAG
instead of UAC, in both cases), to facilitate a
tRNA(Tyr)(CUA)-tyrosyl-tRNA synthetase pair,
improted from Methanococcus jannaschii, capable of
inerting pBpa into the polypeptide. Omega tyrosine
residue was mutated in the same manner in the
Green Fluorescent Protein, which was attached to
the construct as a control. If mutated GFPΩ
maintains its function, we can assume that this
mutation does not greatly disturb Sup35.
2. Primers with TAG
mutation are inserted
3. Polymerase Chain
Reaction (PCR)
5. Incorporation of the single strand
mutation into E. coli, plus ligation
4. Digestion of the
original plasmid
Scheme 1: Site-directed mutagenesis
Figure 1: (a)BpaRS aminoacylating
tRNABpa
CUA and (b)tRNA incorporating
pBpa during translation
pBpa
A U C
U A G
messenger RNA
Ribosome
growing polypeptide a)
b)
tRNA (M. jannaschii)
OR +( )
Scheme 3: Plasmid selection for protein expression
1 2 3 4
IPTG X √ √ √
pBpa √ √ X √
Cam (0.5x) √ √ √ X
a) Sup35(40) GFPΩ,
-CAM
b) Sup35(40) GFPΩ
-pBpa
c) Sup35(40) GFPΩ,
induced
a) Sup35(80) GFPΩ,
-CAM
b) Sup35(80) GFPΩ
-pBpa
c) Sup35(80) GFPΩ,
induced
Figure 4: Sup35(1-40) GFPΩ functionality Figure 5: Sup35(1-80) GFPΩ functionality
• From our results: lane 1 shows uninduced cells,
on all gels. No Sup35 band can be seen.
• Lane 2: induced Sup35-containing cell lysate,
shows bands at expected positions (1-40:
~33kDa, 1-80 ~38 kDa). Full length Sup35
fragments are annotated with a red line.
-In all but 1 case(Sup35(40) GFP), a band
can be seen corresponding to lane 4, i.e.
“truncated” polypeptide. Sup35 (80) should show
a similar pattern to (40), as the “truncated” –
early termination – line should be visible. We
cannot tell why that is not the case. This band
should only be seen in GFP mutants, as Sup35
mutants forming truncated peptides are too
short to be visualized here (<10 kDa).
• Lane 3, E. coli grown without pBpa. They show no
Sup35 presence, similarly to uninduced samples,
except for slightly lower bands. Additionally, GFPΩ
show truncated GFP bands. Due to the lack of pBpa,
translation ends, producing ~30kDa (40) and ~35kDa
(80) truncated peptides. Seen next to the green line.
• Lane 4, without Cam, selective pressure is gone and
Amp resistant bacteria grow preferentially. This
provides a negative control for GFPΩ.
Summary
• We’ve incorporated pBpa into Sup35(1-40) and (1-80).
• We’ve shown that pBpa can be incorporated into another protein –
GFP, while maintaining GFP’s function.
• Sup35 can be mutated at multiple positions.
• We’ve shown that pBpa doesn’t have a great impact on GFP
stability. Assuming that a peripheral tyrosine region of Sup35 is
mutated, it should not disturb protein’s conformation.
• We could use the same mutants for other artificial amino acids, eg.
p-iodo-l-phenylalanine, which could be visualized through Cryo EM.
• If we were to continue with pBpa, we could use UV and mass
spectrometry to detect where exactly the residues are, in relation
to other amino acids.
~180 kDa
~130
~70
~55
~40
~35
~25
~15
~10
Induced Sup35(1-40)
Induced Sup35(1-80)
~100
Figure 6: Original Sup35, induced not purified
1 2 3 4
Sup35(80) Y49
1 2 3 4
Sup35(80) GFPΩ
1 2 3 4
Mutation : Sup35(40) GFPΩ
1 2 3 4
Sup35(40) Y13
1 2 3 4
Sup35(80) Y13
Figure 7: SDS-PAGE of mutated Sup35 strands
~10
~15
~25
~35
~40
~55
~70
~100
~130
~180 kDa
~10
~15
~25
~35
~40
~55
~70
100 kDa
~10
~15
~25
~35
~40
~55
~70
~100 kDa
~10
~15
~25
~35
~40
~55
~70
~100
~130
~180 kDa
~10
~15
~25
~35
~40
~55
~70
~100
~130
~180 kDa
Scheme 2: Site-directed mutagenesis
Figure 3: pBpa structure