PAPERS, PRE-PRINTS, REVIEWS
1. Papers, Pre-prints, Reviews
Multiviral Quartet Nanocages Elicit Broad Anti-Coronavirus Responses for Proactive Vaccinology
Rory A. Hills, Tiong Kit Tan, Alexander A. Cohen, Jennifer R. Keeffe, Anthony H. Keeble, Priyanthi N.P. Gnanapragasam, Kaya N.
Storm, Michelle L. Hill, Sai Liu, Javier Gilbert-Jaramillo, Madeeha Afzal, Amy Napier, William S. James, Pamela J. Bjorkman, Alain R.
Townsend and Mark Howarth.
bioRxiv 2023 Feb PDF+SI Abstract
There are many pathogens, particularly in bats, with the potential to infect human
cells. Here we explore how to produce scalable vaccines with broad protection, so that
one can start to prepare vaccines in advance of a related pathogen emerging as a
Structural basis for a conserved neutralization epitope on the receptor-binding domain of SARS-CoV-2.
Kuan-Ying Huang, Xiaorui Chen, Arpita Mohapatra, Vy H. T. Nguyen, Lisa Schimanski, Tiong Kit Tan, Pramila Rijal, Susan Vester, Rory
Hills, Mark Howarth, Jennifer Keeffe, Alexander Cohen, Leesa Kakutani, Yi-min Wu, Md. Shahed-Al-Mahmud, Yu-Chi Chou, Pamela
Bjorkman, Alain Townsend, Che Ma.
Nature Communications Jan 2023 19;14(1):311 PDF+SI Abstract
New variants of SARS-CoV-2 are resistant to antibodies from existing vaccines or therapeutic antibody
cocktails. By studying human immune responses, we identify an antibody that “gets under the skin” of
the virus, achieving broad recognition by binding to protein features normally buried.
CD4-binding site immunogens elicit heterologous anti-HIV-1 neutralizing antibodies in transgenic and wildtype animals
Gristick HB, Hartweger H, Loewe M, van Schooten J, Ramos V, Oliveira TY, Nishimura Y, Koranda NS, Wall A, Yao KH, Poston D,
Gazumyan A, Wiatr M, Horning M, Keeffe JR, Hoffmann MAG, Yang Z, Abernathy ME, Dam KA, Gao H, Gnanapragasam PNP,
Kakutani LM, Pavlovitch-Bedzyk AJ, Seaman MS, Howarth M, McGuire AT, Stamatatos L, Martin MA, West AP Jr, Nussenzweig MC,
Science Immunology. 2023 Feb 17;8(80):eade6364 PDF+SI Abstract
It is very difficult to get an HIV vaccine that
protects against a wide range of strains. The
CD4 binding site on HIV’s envelope protein
is a good location for binding of antibodies
that neutralize diverse strains. Here the
envelope protein was evolved to be better
at inducing such antibodies, towards a
more protective vaccine.
SpySwitch enables pH- or heat-responsive captureand release for plug-and-display nanoassembly
Susan K. Vester, Rolle Rahikainen, Irsyad N. A. Khairil Anuar, Rory A. Hills, Tiong Kit Tan and Mark Howarth.
Nature Communications 2022 June 13:3714 PDF+SI Abstract
SpyTag technology has become established
as a powerful tool for vaccine assembly.
Here we design/evolve a switchable
SpyCatcher to be a valuable approach for
purification under mild conditions with high
purity. We also find a patient-derived
antibody has exceptional breadth of
recognition across related bat viruses to
Modular capsid decoration boosts adenovirus vaccine-induced humoral and cellular immunity against SARS-CoV-2
Matthew D. J. Dicks, Louisa M. Rose, Lesley A. H. Bowman, Carl Graham, Katie J. Doores, Michael H. Malim, Simon J. Draper, Mark
Howarth, Sumi Biswas
Molecular Therapy 2022 Aug PDF+SI Abstract
Adenoviruses are important vectors for vaccines and cancer immunotherapy.
They are particularly good at inducing cytotoxic T cell responses. This study
shows how to covalently decorate the adeno surface to enhance the
induction of antibody responses. This display also can shield from antibodies
against the adenovirus, which may increase the possibility to use the same
vector again and again.
Simultaneous identification of viruses and SARS-CoV-2 variants with programmable DNA nanobait
Filip Bošković, Jinbo Zhu, Ran Tivony, Alexander Ohmann, Kaikai Chen, Mohammed F. Alawami, Milan Đorđević, Niklas Ermann,
Joana Pereira Dias, Michael Fairhead, Mark Howarth, Stephen Baker, Ulrich F. Keyser
Nature Nanotechnology 2023 PDF+SI
Virus-like particles against infectious disease and cancer: guidance for the nanoarchitect
Rory A. Hills and Mark Howarth
Current Opinion in Biotechnology 2021 Oct 29;73:346-354 Full text Abstract
How recent advances in nanoassembly, adjuvants, conjugation
have helped to establish principles for designing vaccines and
Gastrobodies are engineered antibody mimetics resilient to pepsin and hydrochloric acid
Niels Wicke, Michael R. Bedford, Mark Howarth
Communications Biology 2021 Aug 11;4(1):960 PDF+SI Abstract
Wouldn’t it be good if protein therapeutics could be eaten? Here we
generate a selectable protein scaffold by evolutionary and
computational engineering of a plant inhibitor, towards this goal. We
show that our binder to a C. difficile toxin is resilient to pH 1 + pepsin, as
well as testing stability to other proteases and bile acids.
DogCatcher allows loop-friendly protein-protein ligation
Anthony H. Keeble, Vikash K. Yadav, Matteo P. Ferla, Claudia C. Bauer, Eulashini Chuntharpursat-Bon, Jin Huang, Robin S. Bon and
Cell Chemical Biology 2022 Feb 17;29(2):339-350. PDF + SI Abstract
For molecular assembly (vaccines, enzymes etc.) it is often
good to lock on through a loop rather than a terminus. We
evolved the DogCatcher protein to be the fastest way to
do such a reaction.
NeissLock provides an inducible protein anhydride for covalent targeting of endogenous proteins
Arne H. A. Scheu*, Sheryl Y. T. Lim*, Felix J. Metzner, Shabaz Mohammed, and Mark Howarth
Nature Communications 2021 12:717 * joint first author PDF + SI Abstract
Inspired by the surface chemistry of Neisseria meningitidis, we harness a
genetically-encoded anhydride to trap protein-protein interactions. This
may be useful for decoration of a range of therapeutic modalities.
Neutralization of SARS-CoV-2 and zoonotic coronavirus threats by mosaic nanoparticle vaccination
Alexander A Cohen, Priyanthi N.P. Gnanapragasam, Yu E. Lee, Susan Ou, Leesa M. Kakutani, Jennifer R Keeffe, Christopher O
Barnes, Hung-Jen Wu, Mark Howarth, Anthony P West, Michel C. Nussenzweig, Pamela J Bjorkman.
Science 12 Jan 2021 PDF+SI Abstract
Various coronaviruses have the potential to cause human
outbreaks. Display of RBD mixtures on the same virus-like
particle induces antibodies that can neutralize multiple
A COVID-19 vaccine candidate using SpyCatcher multimerization of the SARS-CoV-2 spike protein receptor-binding domain
induces potent neutralising antibody responses
Tiong Kit Tan*, Pramila Rijal, Rolle Rahikainen, Anthony Keeble…Mark Howarth*, Alain Townsend*.
Nature Communications 2021 12: 542 PDF+SI Abstract *corresponding authors
Immunization of different animal models, indicating a strong
antibody response to multiple sites on RBD, which should be
hard for virus mutants to evade.
Overcoming Symmetry Mismatch in Vaccine Nanoassembly via Spontaneous Amidation
Rolle Rahikainen, Pramila Rijal, Tiong Kit Tan, Hung-Jen Wu , Anne-Marie C. Andersson, Jordan R. Barrett, Thomas A. Bowden,
Simon J. Draper, Alain R. Townsend, Mark Howarth
Angewandte Chemie 2020 PDF+SI Abstract
Nanoassembly can play a key role in generating effective vaccines for emerging
(SARS-CoV-2) and established (Influenza) pandemic threats. Spy-display
allowed the nanocage to adapt to the challenge of bacterial or viral antigens
with diverse cyclic and dihedral symmetries. Establishing display of both
trimeric and tetrameric antigens may lead to an Influenza vaccine with broader
Power to the Protein: Enhancing and Combining Activities using the Spy Toolbox
Anthony H. Keeble and Mark Howarth
Chemical Science 2020 11, 7281 - 7291 PDF Abstract
Review bringing together the different possibilities arising from precise
unbreakable ligation for imaging, nanotechnology, enzymes etc.
Transmembrane protein rotaxanes reveal kinetic traps in the refolding of translocated substrates
Jianfei Feng, Pablo Martin-Baniandres, Michael J. Booth, Gianluca Veggiani, Mark Howarth, Hagan Bayley and David Rodriguez-
Communications Biology, 2020 Apr 3;3(1):159. PDF+SI Abstract on Medline
Nanopores have produced a revolution in the sequencing of
DNA. This paper develops their ability to be used for protein
SnoopLigase-Mediated Peptide-Peptide Conjugation and Purification.
Buldun CM, Khairil Anuar INA, Howarth M.
Methods Mol Biol. 2021;2208:13-31. Abstract on Medline
Approaching infinite affinity through engineering of peptide–protein interaction
Anthony H. Keeble, Paula Turkki, Samuel Stokes, Irsyad N. A. Khairil Anuar, Rolle Rahikainen, Vesa P. Hytönen and Mark Howarth
PNAS 2019 December 26, 116 (52) 26523-26533. PDF+SI Abstract on Medline
Design through computation and evolution of an interaction
forming an amide bond with kinetics near the diffusion limit.
We explore the system by HDX mass spectrometry, stop-flow
FRET, flow cytometry and reconstitution of cellular
mechanosensing. (See note on codon usage at Reagents
Spy&Go purification of SpyTag-proteins using pseudo-SpyCatcher to access an oligomerization toolbox
Irsyad N. A. Khairil Anuar, Anusuya Banerjee, Anthony H. Keeble, Alberto Carella, Georgi I. Nikov & Mark Howarth
Nature Communications 2019 volume 10, Article number: 1734 PDF+SI Abstract on Medline
A new route for affinity purification + platforms to plug in
dimer to heptamer assembly, applied to activating
cancer cell apoptosis. See our SpySwitch paper above for further optimized
SnoopLigase peptide-peptide conjugation enables modular vaccine assembly
Anne-Marie C. Andersson, Can M. Buldun, David J. Pattinson, Simon J. Draper & Mark Howarth
Sci Rep 2019 March, 9:4625 PDF+SI
Abstract on Medline
Covalent assembly for immunization, applied to
enhance immune responses, particularly for
malaria antigens or cancer neoepitopes.
Insider information on successful covalent protein coupling with help from SpyBank
Keeble AH, Howarth M.
Methods in Enzymology 2019;617:443-461. PDF Abstract on Medline SpyInfo page
Advice and survey on using SpyTag in
various applications. (See note on codon usage at Reagents page).
Assembling and decorating hyaluronan hydrogels with twin protein superglues to mimic cell-cell interactions
Wieduwild R, Howarth M.
Biomaterials. 2018 Jul 17;180:253-264. PDF Abstract on Medline
Modular decoration of extracellular matrix in 3D
cell culture to explore how adhesion signals program
Engineering a Rugged Nanoscaffold To Enhance Plug-and-Display Vaccination.
Bruun TUJ*, Andersson AC*, Draper SJ, Howarth M.
ACS Nano. 2018 Jul 26. doi: 10.1021/acsnano.8b02805. PDF+SI Abstract on Medline *joint first author
Rational enhancement of computationally-
designed nanoparticle so easy to increase
antigen immunogenicity, for malaria and
other vaccine challenges.
Nanobiotechnology: Baby steps and giant strides towards molecular mastery.
Howarth M, Jaramillo A.
Current Opinion in Biotechnology. 2018 Jun;51:iv-vi. doi: 10.1016/j.copbio.2018.05.001.
New routes and opportunities for modular construction of particulate vaccines: stick, click and glue.
Brune KD, Howarth M.
Frontiers in Immunology 2018 Jun 26;9:1432. PDF Abstract on Medline
Including personalized cancer vaccines, rapid response
to pandemic threats and in-field vaccine assembly.
SnoopLigase catalyzes peptide-peptide locking and enables solid-phase conjugate isolation.
Buldun CM*, Jean JX*, Bedford MR, Howarth M.
Journal of the American Chemical Society. 2018 Feb 28;140(8):3008-3018. PDF+SI Abstract on Medline *joint first author
Rational and computational design of a new enzyme
+ its activity in making other enzymes super-resilient.
Nanoteamwork: covalent protein assembly beyond duets towards protein ensembles and orchestras.
Banerjee A and Howarth M.
Current Opinion in Biotechnology 2017 Nov 21;51:16-23 PDF Abstract on Medline
Analyzing progress and concepts for building cooperative
and harmonious teams in synthetic biology.
Amine Landscaping to Maximize Protein-Dye Fluorescence and Ultrastable Protein-Ligand Interaction.
Jacobsen MT, Fairhead M, Fogelstrand P, Howarth M.
Cell Chemical Biology 2017 Aug 17;24(8):1040-10 PDF Abstract on Medline
Optimizing one of the most common
protein modifications, NHS on
amines, to generate Flavidin,
fluorophore-friendly streptavidin for
Evolving accelerated amidation by SpyTag/SpyCatcher to analyze membrane dynamics.
Keeble AH, Banerjee A, Ferla MP, Reddington SC, Khairil Anuar INA, Howarth M.
Angewandte Chemie 2017 Dec 22;56(52):16521–16525 PDF Abstract on Medline Other papers/patents using SpyTag
Phage display evolution of our bacterial superglue,
allowing irreversible fluorescent tracking of an
enterohaemorrhagic platform at the bacterial outer
Smart superglue in streptococci? The proof is in the pulling
Journal of Biological Chemistry 2017 May 26;292(21):8998-8999 PDF Abstract on Medline
News and Views on mechanical proof-reading in
Dual plug-and-display synthetic assembly using orthogonal reactive proteins for twin antigen immunization
Brune KD, Buldun CM, Li Y, Taylor IJ, Brod F, Biswas S, Howarth M.
Bioconjugate Chemistry 2017 May 17;28(5):1544-1551 PDF Abstract on Medline
Accelerating vaccine development for multi-strain
or multi-disease protection.
Extracellular self-assembly of functional and tunable protein conjugates from Bacillus subtilis
Gilbert C, Howarth M, Harwood CR, Ellis T.
ACS Synthetic Biology 2017 Jun 16;6(6):957-967 PDF Abstract on Medline
SpyRing enzyme stabilization (for xylanase, relevant
to food, paper, biofuel), assembled by one cell or
by cell cooperation.
Nanoassembly routes stimulate conflicting antibody quantity and quality for transmission blocking malaria vaccines.
Leneghan DB, Miura K, Taylor IJ, Li Y, Jin J, Brune KD, Bachmann MF, Howarth M, Long CA, Biswas S.
Sci Rep 2017 7:3811 PDF Abstract on Medline
Pushing for the best immunogen, how antibody titre and activity differ
in stopping mosquitoes getting infected.
Controlling multivalent binding through surface chemistry: model study on streptavidin
Dubacheva GV, Araya C, Fairhead M, Codée J, Howarth M, Richter RP.
Journal of the American Chemical Society 2017 Mar 22;139(11):4157-4167 PDF Full text Abstract on Medline
Streptavidin can steal biotinylated lipids from membranes
A single molecule assay to probe monovalent and multivalent bonds between hyaluronan
and its key leukocyte receptor CD44 under force
Bano F, Banerji S, Howarth M, Jackson DG, Richter RP.
Sci Rep 2016 Sep 29;6:34176 PDF+SI Abstract on Medline
Understanding extracellular matrix interaction for recruiting
immune cells and anchoring circulating tumour cells
Programmable polyproteams built using twin peptide superglues
Veggiani G, Nakamura T, Brenner MD, Gayet RV, Yan J, Robinson CV, Howarth M.
PNAS 2016 Feb 2;113(5):1202-7 PDF+SI Abstract on Medline
A platform to build molecular teams,
here applied to optimize cancer cell
killing, combining ligation of a Death
Receptor with Growth Factor receptors.
Plug-and-Display: decoration of Virus-Like Particles via isopeptide bonds for modular immunization
Brune KD, Leneghan DB, Brian IJ, Ishizuka AS, Bachmann MF, Draper SJ, Biswas S, Howarth M.
Sci Rep 2016 Jan 19;6:19234 PDF+SI Press release Abstract on Medline
A strategy to accelerate vaccine generation, which could have broad application
for cancer immunotherapy and infectious diseases.
SpyRings Declassified: A Blueprint for Using Isopeptide-Mediated Cyclization to Enhance Enzyme Thermal Resilience
C. Schoene, S.P. Bennett, M. Howarth.
Methods in Enzymology 2016;580:149-67 PDF Abstract on Medline
SpyRing interrogation: analyzing how enzyme resilience can be achieved with phytase and distinct cyclization chemistries
Schoene C, Bennett SP, Howarth M.
Sci Rep 2016 Feb 10;6:21151 PDF+SI Abstract on Medline
Understanding a generic approach for enhancing
Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher
Reddington SC, Howarth M.
Current Opinion in Chemical Biology 2015 Oct 27;29:94-99 PDF Abstract on Medline
Say it with proteins: an alphabet of crystal structures
Nature Structural & Molecular Biology 2015 May;22(5):349 PDF For video, files to download and more information
Happy that >1000 views on YouTube, but
a while to go before into Justin Bieber territory.
Site-Specific Biotinylation of Purified Proteins Using BirA
Fairhead M, Howarth M.
Methods in Molecular Biology 2015;1266:171-84 PDF Abstract on Medline
SpyLigase peptide–peptide ligation polymerizes affibodies to enhance magnetic cancer cell capture
Fierer J.O.*, Veggiani G.*, Howarth M.
PNAS 2014;111(13):E1176-81 *joint first author PDF+SI Abstract on Medline
Developing a new way to irreversibly lock two peptides
(via a 3-way split protein) to assemble tentacle-like
polymers of affibodies/antibodies for improving
sensitivity of isolation of cancerous cells.
SpyAvidin Hubs Enable Precise and Ultrastable Orthogonal Nanoassembly
Fairhead M, Veggiani G, Lever M, Yan J, Mesner D, Robinson CV, Dushek O, van der Merwe PA, Howarth M.
Journal of the American Chemical Society 2014;136(35):12355-63
PDF Supporting Info. Abstract on Medline Robinson Lab van der Merwe lab
Combining one of the best interactions
in nature with our bacterial superglue.
SpyAvidins should help detection of
weak interactions, such as for finding
anti-cancer T cells.
Superglue from Bacteria: Unbreakable Bridges for Protein Nanotechnology
Veggiani G., Zakeri B., Howarth M.
Trends in Biotechnology 2014 Oct;32(10):506-12 PDF Abstract on Medline
Love-Hate ligands for high resolution analysis of strain in ultra-stable protein:small molecule interaction
Fairhead M, Shen D, Chan LK, Lowe ED, Donohoe TJ, Howarth M.
Bioorganic & Medicinal Chemistry 2014 Oct 1;22(19):5476-86
PDF Supporting Info. Abstract on Medline Donohoe lab PDB entries: SA-LH1, SA-LH3, SA-LH4, A86D-LH4
A novel chemical biology approach
to understand how force distorts
SpyTag/SpyCatcher Cyclization Confers Resilience to Boiling on a Mesophilic Enzyme
Schoene C., Fierer J.O., Bennett S.P., Howarth M.
Angewandte Chemie 2014 Jun 10;53(24):6101-4.
PDF+SI Abstract on Medline BBSRC News story
Towards a general way to make enzymes that can tolerate
tough conditions (e.g. for diagnostics or for biofuels), we
found that locking the tails together with our bacterial
superglue gave dramatic increases in resilience.
Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag
Li L., Fierer J.O., Rapoport T.A., Howarth M.
Journal of Molecular Biology 2014 Jan 23;426(2):309-317
PDF Supporting Info. Abstract on Medline PDB entries: SpyTag/SpyCatcher, SpyTag/SpyCatcherΔN1
Crystal structure of the SpyCatcher/SpyTag complex,
a genetically encoded peptide forming a spontaneous
isopeptide bond to its protein partner, and minimization
of the size of SpyCatcher.
Plug-And-Play Pairing Via Defined Divalent Streptavidins
Fairhead M, Krndija D, Lowe ED, Howarth M.
Journal of Molecular Biology 2014 Jan 9;426(1):199-214
PDF Abstract on Medline PDB entries: cis-divalent, trans-divalent, trans-divalent with biotin-fluorescein
Streptavidin is one of the most widely
used building blocks in biology and
also has given promising results for
improving delivery of cancer radiotherapy.
We developed charged tags to enable
isolation of precise tetramers for robust
nanoassembly and validated the structures
Cholesterol loading and ultrastable protein interactions determine the level of tumor marker required for optimal
isolation of cancer cells.
Jain J, Veggiani G, Howarth M.
Cancer Research 2013 Apr 1;73(7):2310-21 PDF Abstract on Medline Supporting Info.
Detecting the circulating tumor cells in blood
samples is one of the most promising ways to
achieve early cancer diagnosis and rapid
feedback on cancer treatment. Here we
establish the key factors for magnetic cancer
cell isolation and use this insight to enhance
the isolation of cells expressing low levels of
cancer marker. Described for a general
audience on the Oxford University Science Blog.
Hydroxy-Terminated Conjugated Polymer Nanoparticles Have Near-Unity Bright Fraction and Reveal Cholesterol-
Dependence of IGF1R Nanodomains.
Koner AL, Krndija D, Hou Q, Sherratt DJ, Howarth M.
ACS Nano 2013 Feb 26;7(2):1137-44 PDF Abstract on Medline Supporting Info.
Single molecule imaging is a powerful way to gain insight
into receptor function. Here we show that Conjugated
Polymer Nanoparticles may be the best nanoparticles
for single molecule imaging, because, unlike quantum
dots, these particles do not blink and there are almost
no "ghost" particles which fail to emit.
Quantum Dot Targeting with Lipoic Acid Ligase and HaloTag for Single Molecule Imaging on Living Cells.
Liu DS, Phipps WS, Loh KH, Howarth M, Ting AY.
ACS Nano 2012 Dec 21;6(12):11080-7. PDF Abstract on Medline Supporting Info.
Using two re-designed enzymes, a covalent connection can
be made between a peptide-tagged cell surface protein and
fluorescent nanoparticle. This enabled two different types of
adhesion molecules to be followed at the single molecule
level on living cells.
Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin.
Zakeri B*, Fierer JO*, Celik E, Chittock EC, Schwarz-Linek U, Moy VT, Howarth M.
PNAS 2012 Mar 20;109(12):E690-7. PDF Abstract on Medline Supporting Info. *joint first author
Graphic design with Myoungshin Kim
Peptide interactions with proteins are usually weak. Here we show
how to get a genetically encoded peptide to form an irreversible
covalent bond when it binds its protein partner (SpyTag with
SpyCatcher). This may be useful as a cellular padlock, for imaging and
to enable new protein architectures. (Plasmids available from Addgene
repository or directly from us.) Described for a general audience on
the Oxford University Science Blog or by Bijan in a TED talk.
Other papers/patents using SpyTag
A peptide filtering relation quantifies MHC class I peptide optimization.
Dalchau N, Phillips A, Goldstein LD, Howarth M, Cardelli L, Elliott T, Werner JM.
PLoS Computational Biology 2011 Oct;7(10):e1002144. PDF Abstract on Medline Supporting Info.
Collaboration with Microsoft and my previous lab in Southampton to model
mathematically all the steps in peptide presentation for T cell recognition.
Mechanisms for size-dependent protein segregation at immune synapses assessed with molecular rulers.
Alakoskela JM, Koner AL, Rudnicka D, Köhler K, Howarth M, Davis DM.
Biophysical Journal 2011 Jun 22;100(12):2865-74. PDF Abstract on Medline Supporting Info.
The exact intermembrane spacing when a natural killer cell encounters a target cell
(virally infected or cancerous) is important in triggering killer cell activation. In
collaboration with Dan Davis' lab we developed a controlled size-series of fluorescent
proteins and nanoparticles for real-time imaging of how molecules above a certain size
are excluded from the synapse.
How the biotin-streptavidin interaction was made even stronger: investigation
via crystallography and a chimaeric tetramer.
Chivers CE, Koner AL, Lowe ED, Howarth M.
Biochemical Journal 2011 Apr 1;435(1):55-63.
PDF Abstract on Medline PDB entries: apo-traptavidin biotin-traptavidin
Our first foray into structural biology, to help determine the origin of one of the strongest non-covalent
interactions in nature.
The type I IGF receptor translocates to the nucleus of human tumor cells.
Aleksic T, Chitnis M, Perestenko O, Gao S, Thomas P, Turner G, Protheroe A, Howarth M, Macaulay V.
Cancer Research 2010 Aug 15;70(16):6412-9. PDF Abstract on Medline Supporting Info.
The classic model of growth factor receptor signaling is that
the activated receptor at the plasma membrane/endosomes,
activates down-stream pathways. Components of these
pathways then enter the nucleus, leading to changes in gene
expression. This paper contributes to a new model where the
receptor itself moves to the nucleus and has direct effects on
transcription. We show that nuclear IGF1R is associated with a
more aggressive cancer, suggesting that approaches to block
nuclear translocation could improve survival.
A streptavidin variant with slower biotin dissociation and increased mechanostability.
Chivers CE, Crozat E, Chu C, Moy VT, Sherratt DJ, Howarth M.
Nature Methods 2010 May;7(5):391-93. PDF Abstract on Medline Supporting Info.
Streptavidin binds to the vitamin biotin with one of the
strongest non-covalent interactions in nature. We developed a
mutant of streptavidin, termed traptavidin, which binds biotin
10-times better. We tested traptavidin, by setting up a
molecular car-crash to investigate a motor protein involved in
chromosome segregation. (Traptavidin protein and plasmids
Separating speed and ability to displace roadblocks during DNA translocation by FtsK.
Crozat E, Meglio A, Allemand JF, Chivers CE, Howarth M, Vénien-Bryan C, Grainge I, Sherratt DJ.
EMBO Journal 2010 Apr 21;29(8):1423-33.
PDF Abstract on Medline Supporting Info. Sherratt lab Grainge lab
FtsK is a bacterial motor protein that pumps DNA, so that each daughter cell receives
one copy of the chromosome. In collaboration with the Sherratt lab we explored
through crash-testing how the different active sites in a ring coordinate their firing.
Spontaneous Intermolecular Amide Bond Formation between Side Chains for Irreversible Peptide Targeting.
Zakeri B & Howarth M.
Journal of the American Chemical Society 2010 Apr 7;132(13):4526-7.
PDF Abstract on Medline Supporting Info.
Proof of principle for spontaneous covalent bond formation to a
peptide, developed from a pilin and based on Lysine reaction with
Asparagine. See also our PNAS 2012 paper.
Electrophilic affibodies forming covalent bonds to protein targets.
Holm L, Moody P, Howarth M.
Journal of Biological Chemistry 2009 Nov;284(47):32906-13 PDF Abstract on Medline Supporting Info.
Affibodies are like antibodies but smaller and easier to make. We introduced an
artificial reactive group next to the target binding site of an affibody. This enabled the
affibody to form a specific covalent bond to its target. By preventing affibody
dissociation we could improve detection sensitivity and stability. We are working to
make this reaction faster and more general, to enable detection of disease markers
present in blood at concentrations too low for current tests.
Monovalent, reduced-size quantum dots for imaging receptors on living cells.
Howarth M, Liu W, Puthenveetil S, Zheng Y, Marshall LF, Schmidt MM, Wittrup KD, Bawendi MG, Ting AY.
Nature Methods 2008 May;5(5):397-99. PDF Abstract on Medline
Quantum dots are ultra-bright nanoparticles, which make single
molecule imaging routine. A major problem in the field is that one
nanoparticle can bind several copies of its target receptor. This stops
the receptor moving freely and often activates cell signalling. Here we
develop a way to purify quantum dots with precisely defined numbers
of proteins attached. We use these non-crosslinking high affinity QDs
to image the low density lipoprotein receptor and the tumour marker
Compact Biocompatible Quantum Dots Functionalized for Cellular Imaging.
Liu W, Howarth M, Greytak AB, Zheng Y, Nocera DG, Ting AY, Bawendi MG.
Journal of the American Chemical Society 2008 Jan 30;130(4):1274-84.
PDF Abstract on Medline Supporting Info. Bawendi lab
It has been challenging to reduce quantum dot size at the same time as maintaining high
stability and specificity in their targeting. In this paper we engineer the QD passivating layer
to address this challenge and apply these QDs to image the epidermal growth factor
receptor, an important regulator of cancer cell division.
Tapasin shapes immunodominance hierarchies according to the kinetic stability of peptide-MHC class I complexes.
Thirdborough SM, Roddick JS, Radcliffe JN, Howarth M, Stevenson FK, Elliott T.
European Journal of Immunology 2008 Jan 14;38(2):364-369. PDF Abstract on Medline
A development of work from my DPhil (see PNAS 2004 below), showing that tapasin not
only changes how well peptides are displayed by MHC class I at the cell surface, but also
how well these peptides activate the immune system from DNA vaccination.
Protein imaging in live mammalian cells by site-specific labeling with biotin ligase and monovalent streptavidin.
Howarth M, Ting AY.
Nature Protocols 2008;3(3):534-45. PDF Abstract on Medline
Step-by-step instructions and trouble-shooting guide, for those applying the approaches
we developed for stable cell labeling with biophysical probes and expression of a
streptavidin with a single high affinity binding site. (Plasmids for BirA and monovalent
streptavidin protein available.) Note in our current protocol we use 5 min with a stir-bar
to resuspend inclusion bodies and reduce pipetting effort, while our J Mol Biol paper
2014 describes an easier chromatographic separation.
Giving cells a new sugar-coating.
Howarth M, and Ting AY.
Nature Chemical Biology 2006 Mar;2(3):127-8 PDF Abstract on Medline
News and Views article on a paper by Kevin Yarema feeding cells a thiol sugar. This enables them to redecorate the cell surface
with sialic acids bearing thiols, which changed adhesion of stem cells. We explain how this method works and discuss the
importance of manipulating protein glycosylation.
A monovalent streptavidin with a single femtomolar biotin binding site.
Howarth M, Chinnapen DJ-F, Gerrow K., Dorrestein PC, Grandy MR, Kelleher NL, El-Husseini A, Ting AY.
Nature Methods 2006, Apr;3(4):267-73
PDF Abstract on Medline News and Views Supp. Info. Dorrestein lab
Streptavidin is used in almost every biology lab and also by many physicists and engineering
seeking to manipulate molecules on the nanometre scale. Streptavidin is used so much in
biotechnology because of its rapid, selective and stable binding to anything modified with biotin.
However, streptavidin has 4 binding sites. This can cross-link proteins on the surface of cells and
limit one's control in nanotechnology applications. We made a version where one can control the
number of binding sites from 0-4 but keep the tight binding. Our J Mol Biol paper 2014 describes
an easier chromatographic separation of the monovalent and divalent forms. (Monovalent
streptavidin protein and plasmids available.)
Targeting quantum dots to surface proteins in living cells with biotin ligase.
Howarth M, Takao K, Hayashi Y, and Ting AY.
PNAS 2005, May;102(21):7583-8 PDF Abstract on Medline Supp. Info./Movie Hayashi Lab
Quantum dots are inorganic nanoparticles that are so bright that they make it routine to image single proteins
moving in living cells. Here we develop the use of biotin ligase to add biotin to neurotransmitter receptors on the
neuron surface and then track the receptors with quantum dots.
Site-Specific Labeling of Cell Surface Proteins with Biophysical Probes using Biotin Ligase.
Chen I, Howarth M, Lin W, Ting AY.
Nature Methods 2005 Feb;2(2):99-104 PDF Abstract on Medline Supp. Info.
It is a great challenge to label proteins with new tags that will
probe their function. Biotin ligase recognizes a 15 amino acid
tag specifically over other cytosolic and cell surface proteins.
We get Biotin ligase to add a ketone analog of biotin to a
tagged cell-surface protein. Since there are no ketones on the
cell-surface, this enables us to react the ketone with any
hydrazide-labeled biophysical probe.
DNA Transfection Screening from Single Beads.
Yingyongnarongkul BE, Howarth M, Elliott T, Bradley M.
Journal of Combinatorial Chemistry 2004 Sep-Oct;6(5):753-60.
PDF Supporting info. Abstract on Medline Mark Bradley lab
Solid-phase synthesis of 89 polyamine-based cationic lipids for DNA delivery to mammalian cells.
Yingyongnarongkul BE, Howarth M, Elliott T, Bradley M.
Chemistry 2004 Jan 23;10(2):463-73. PDF Supporting info. Abstract on Medline
One of the greatest challenges in medicine is to get DNA expressed long
term in a wide number of cells, for gene therapy. Even in the laboratory it
is still a challenge to get DNA into many cell-types. However, it is hard to
predict which compound will be best at introducing DNA into cells, known
as DNA transfection. Mark Bradley's group are experts in combinatorial
synthesis, where new compounds are made in a highly parallel fashion,
rather than one at a time. They turned their attention to combinatorial
synthesis of transfection reagents and we helped them in the testing and
design of these reagents. We look at an uncommon class of transfection
agent with only one lipid tail and show that the amount of compound
made on a single polymer bead is sufficient to test transfection activity.
Tapasin enhances MHC class I antigen presentation according to peptide half-life.
Howarth M, Williams A, Tolstrup AB, Elliott T.
PNAS 2004 Aug;101(32):11737-11742 PDF Abstract on Medline Elliott lab
MHC class I presents peptides that are seen by killer T cells and controls
their activation and killing. Tapasin is an ER chaperone that associates with
newly synthesized MHC class I. Here we show that the interaction with
tapasin changes the repertoire of peptides that MHC class I presents, to
favour stable peptides, with consequences for autoimmune disease and
The processing of antigens delivered as DNA vaccines.
Howarth M, Elliott T.
Immunological Reviews 2004 199:27-39 PDF Abstract on Medline
DNA vaccination involves stimulating an immune response with DNA encoding pathogenic genes or gene fragments, rather than
immunizing with protein or attenuated virus, as is more conventional. Trials of DNA vaccination are taking place for many
diseases, including skin cancer and HIV. This review discusses how our basic knowledge of MHC class I and II antigen
presentation may explain DNA vaccination results and guide future strategies.
Assembly and antigen-presenting function of MHC class I molecules in cells lacking the ER chaperone calreticulin.
Gao B, Adhikari R, Howarth M, Nakamura K, Gold MC, Hill AB, Knee R, Michalak M, Elliott T.
Immunity 2002 Jan;16(1):99-109 PDF Abstract on Medline Bin Gao lab
Calreticulin is a multi-talented protein, involved in calcium homeostasis, gene regulation, and folding of secretory proteins. Here
we tested how the presence of calreticulin changed the loading of MHC class I with peptide and its exit from the endoplasmic
Processing and presentation of glycoproteins in the MHC class I and II antigen presentation pathways.
Golgher DB, Elliott T, Howarth M.
Immunobiology of Carbohydrates, chapter, Landes Biosciences, 2002. PDF
Link on Google books (can click through the pages)
MHC class I presents peptides to killer T cells and MHC class II presents peptides to
helper T cells. Many cellular proteins are glycosylated, yet how this affects what peptides
are presented by MHC is little understood and challenging to study. It may have
relevance to immune responses in conditions such as allergy and cancer. We also point
out many of the key questions still unanswered.
The quantity of naturally processed peptides stably bound by HLA-A*0201 is significantly reduced in the absence of
Barber LD, Howarth M, Bowness P, Elliott T.
Tissue Antigens 2001 Dec;58(6):363-8 PDF Abstract on Medline
MHC class I presents a sample of peptides derived from cellular proteins, allowing killer T cells to monitor what is going on inside
the cell. It is possible to profile these peptides by eluting them with acid and then HPLC analysis. Here we see the effect of the ER
chaperone tapasin on the abundance and nature of peptides eluted from the most common HLA allele in Western populations.
Switchable polypeptide and its use for gentle affinity purification. (SpySwitch)
Howarth M, Khairil Anuar I.N.A., Rahikainen R.
2021 UK Intellectual Property Office 2117283.8
Polypeptides that interact with peptide tags at loops or termini and uses thereof. (DogCatcher)
Howarth M, Yadav V, Ferla M.
2021 UK Intellectual Property Office 2104999.4 PDF
Ligand-binding polypeptides and uses thereof. (Gastrobodies)
Howarth M, Wicke N.
2021 UK Intellectual Property Office 2019817.2 PDF
System for covalently linking proteins. (NeissLock)
Howarth M, Scheu AHA, Lim SYT.
2020 UK Intellectual Property Office 2003683.6 PDF
Viruses with modified capsid proteins.
Dicks M, Howarth M, Biswas S.
2019 UK Intellectual Property Office 1915905.2 PDF
Polypeptide with enhanced rate of spontaneous isopeptide bond formation with its peptide tag partner and uses
Howarth M, Keeble A, Carella A.
2019 UK Intellectual Property Office 1903479.2 PDF
Polypeptide and its use in affinity purification. (Spy&Go)
Howarth M, Khairil Anuar I.N.A.
2018 UK Intellectual Property Office 1819850.7 PDF
Peptide ligase and use thereof. (SnoopLigase)
Howarth M, Buldun C.
2017 UK Intellectual Property Office 1705750.6 PDF
Proteins and peptide tags with enhanced rate of spontaneous isopeptide bond formation and uses thereof. (Spy002)
Howarth M, Keeble A.
2017 UK Intellectual Property Office 1706430.4 PDF
Mutant streptavidin with improved fluorescent brightness and uses thereof.
Howarth M, Jacobsen MT.
2016 UK Intellectual Property Office 1616051.7 PDF
Methods and products for fusion protein synthesis
Howarth M, Veggiani G, Gayet R.
2015 UK Intellectual Property Office 1509782.7, United Kingdom Patent application (described by Oxford University Innovation)
Peptide tag systems that spontaneously form an irreversible link to protein partners via isopeptide bonds.
Howarth M, Zakeri B.
2010, UK Intellectual Property Office 1002362.0 for PDF
High Stability Streptavidin Mutant Proteins. (Traptavidin)
Howarth M, Chivers C.
2009, UK Intellectual Property Office 0919102.4 PDF
Controlled modification of semiconductor nanocrystals.
Ting AY, Bawendi MG, Howarth M, Liu W.
2007, US Patent and Trademark Office. for PDF
Monovalent Streptavidin compositions.
Ting AY, Howarth M.
2006, US Patent and Trademark Office. for PDF