2. Lambda Capsid
Assembly
• Lambda is an icosahedral T=7 laevo
bacteriophage.
• The major capsid proteins in lambda are gpE
and gpD.
• 420 copies of gpD are displayed on the surface
of the capsid.
• gpD is important for stabilization of the
prohead after headful packaging of DNA.
• Without gpD, Lambda can only package 82%
of it’s wildtype genome.
Fig 1. Illustration of capsid assembly
For lambda bacteriophage.
3. Properties of gpD
• gpD is an 11.4kDa, 109aa protein
that decorates the capsid at quasi 3
and 6-fold vertices.
• gpD is monomeric in solution, but
forms homotrimers during capsid
assembly.
• Interaction of the first 14 N-terminal
residues with the E-loop of gpE is
critical for gpD binding to the
capsid.
• Both N and C-terminal ends of gpD
direct away from gpD-gpE
interface, making them available for
D-fusions. Fig 2. Lambda bacteriophage
showing association of gpD trimers
around the gpE hexamer asymmetric
subunit.
4. Fig 3. gpD trimers orient
in hexamers around the
gpE asymmetric subunit.
gpD trimers form
“dimples” on the capsid
surface, with each
monomer interacting with
the E loop of gpE.
5. Dual Control
Mechanism in
Phage Display
Fig 4. Illustration of the
infection cycle for
i434Dam123 phage in a
D-fusion expressing
host.
6. D-fusion
Constructs
Fig 5. Temperature-sensitive cI857 will
dissociate from operator sites at elevated
temperatures, relieving repression and
allowing transcription of D-fusion. This
provides regulation of D-fusion for
optimized phage display.
7. Isolating missense
mutants of
i434Dam123
• i434Dam123 has a premature
STOP at codon 95 that prevents
translation of D.
• Using different Sup strains and a
Dam15 mutant, it was shown
that missense mutants beneficial
to phage display could be
isolated (1).
• D+ expression can be regulated
by isolating missense mutations
of Dam123, some of which may
improve phage decoration.
Fig 6. Sequence alignment of DWT and Dam123 showing the
premature stop codon, and possible missense mutations with
corresponding amino acids that could allow bypass of the nonsense
mutation.
Possible
Revertants
9. Selecting for IPDF
Mutants
Fig 8. Selection
scheme for IPDF
mutants.
• IPDF mutants were selected for use as a
background for selecting mutants with
improved plating in the presence of gpD-
fusions.
• i434Dam123 was plated on 594 to select for
revertants.
• Plaques were picked onto 594 and 594[p617] to
select for mutants that had lost the ability to
plate with the gpD-fusion.
• Putative IPDF mutants were consecutively
stabbed onto 594 and 594[p617].
• Primary lysates were prepared from the 594
plate. All mutants except for IPDF D1-t
showed significantly inhibited plating on
594[p617].
10. Fig 9. Selection of IPDF mutants
for preparation of primary lysates.
All IPDF mutants except for IPDF
D1-t showed significantly reduced
plating on 594[p617].
11. IPDF Plating and
Sequencing Results
• Plating results show that
the expression of The N-
terminal D-fusion from
the host inhibits plating of
the IPDF mutants.
• However, sequencing
results show that the
mutation responsible for
the IPDF phenotype is not
present in D or E.
• The IPDF mutants were D
revertants!
Fig 10. IPDF mutant plating data
showing severe plating inhibition
when the D-fusion is expressed.
12. Selecting for
supIPDF Mutants
• IPDF lysates were plated on 594
and 594[p617].
• Plaques present on the 594[p617]
plate were then further stabbed
onto 594[p617] and 594.
Fig 11. Selection scheme
for supIPDF mutants.
13. • supIPDF mutants were passaged
through 3 cycles of plating on
594[p617].
• supIPDF plating on 594 and
594[p617] produced equal and
high titres.
Fig 12. Selection of supIPDF
mutants for primary lysates.
Plating data shows strong
suppression of the IPDF
phenotype.
14. Sequencing Results for
supIPDF Mutants
Fig 13. Sequence alignment for supIPDF mutants and i434Dam123
against Wildtype D (5747bp-6709bp). The results show that all
supIPDF mutants are true revertants of D.
• supIPDF mutants were also wildtype for both D and E.
• The sequencing data suggests that the mutations responsible for both
IPDF and supIPDF phenotypes are in other genes that encode proteins
that interact with gpD or gpE.
15. Conclusions
• The Glutamate at codon 95 for gpD
is critical to gpD function, no other
amino acids were substituted.
• There are other proteins besides
gpD and gpE that affect plating in
the presence of gpD-fusions.
• The frequency that supIPDF
mutants are isolated from IPDF
mutants is variable.
Fig 14.
16. Possible Sources for
the IPDF and
supIPDF Phenotypes
• Because the mutations do not
directly affect D or E, it is
hypothesized that the mutations
are in other genes that interact
with D and E.
• Other possible genes affected by
IPDF/supIPDF mutations: Nu3,
B, C, Fi.
• Different IPDF mutants produced
supIPDF mutants at variable
rates: multiple and different
types of mutations responsible
for the IPDF phenotype?
Fig 15. Protein interaction map
illustrating relationships amongst the
different capsid proteins.
17. Possible Protein
Interactions producing an
IPDF Phenotype
• Possible protein interactions causing an IPDF
phenotype:
1. Changes in proteolytic activity of gpC
affecting lengths or number of X1 and X2 that
are produced.
• This may cause the remaining gpE to
preferentially bind gpD-fusion instead of
gpD.
3. Any of these mutations that cause
preferential binding of gpD-fusion over
gpD will increase capsid instability as a
result of not enough stabilizing gpD
binding the capsid.
Fig 16. Lambda capsid assembly and maturation.
18. Future Experiments
• Use of DΔ lambda for D and D-fusion plating
to generate mosaic Lambda display particles
(LDPs)
• DΔ lambda would be generated by
recombineering out D, and deleting 18% of
the non-essential genome.
• Host strains would have both D-fusion and D
plasmids under the control of cI857.
• The progeny phage would be genetically
unlinked from the phenotype, allowing the
use of LDPs without concern of genetic
transfer into the environment.
Fig 17. Illustration of display
particles generated through
exogenous expression of both
gpD and gpD-fusion from a
host strain.
20. Notes
Fig 3. Produced using figures 1.A, 1.B, and 1.D from Lander, Gabriel C., et al. "Bacteriophage lambda stabilization by auxiliary
protein gpD: timing, location, and mechanism of attachment determined by cryo-EM." Structure 16.9 (2008): 1399-1406.
Produced using Figure 4 from Yang, Fan, et al. "Novel fold and capsid-binding properties of the λ-phage display platform
protein gpD." Nature Structural & Molecular Biology 7.3 (2000): 230-237.
Fig 5. Produced using figure 1.B from Hayes, Sidney, Lakshman NA Gamage, and Connie Hayes. "Dual expression system for
assembling phage lambda display particle (LDP) vaccine to porcine Circovirus 2 (PCV2)." Vaccine 28.41 (2010): 6789-6799.
Fig 6. Wildtype D sequence from Genebank file, GeneID: 2703529, NC_001416.1.
Fig 12. https://s-media-cache-ak0.pinimg.com/originals/ed/9c/25/ed9c25169b13d4897773b39b0b3569ec.jpg
Fig 1 and 16 Produced using figure 4a and b from Dokland, Terje, and Helios Murialdo. "Structural transitions during maturation of
bacteriophage lambda capsids." Journal of molecular biology 233.4 (1993): 682-694.
1. Nicastro, Jessica, et al. "Construction and analysis of a genetically tuneable lytic phage display system." Applied microbiology
and biotechnology 97.17 (2013): 7791-7804.
