Summer 2014
Orchestrated by
The Silver Bullet Machine Manufacturing Company Limited
first draft 16th september
Creativity@home
Re-inventing protein purification
first draft 16th september
A key step in almost every biochemical process is protein purification. And, ever since the early days of
biochemistry, people have designed a variety of processes to help make that happen. These processes are very
widely-known, and extensively used - and they all work, and work well. These processes are of such importance that
several Nobel Prizes have been awarded to their developers – for example, Theodore Svedberg (Chemistry, 1926) for
the ultracentrifuge; James Sumner (Chemistry, 1946) for discovering that enzymes can be crystallised; John
Northrop and Wendell Meredith (also Chemistry, 1946) for preparing enzymes and virus proteins in a pure form; Arne
Tiselius (Chemistry, 1948) for electrophoresis; and Archer Martin and Richard Synge (Chemistry, 1952) for partition
chromatography. So, surely, everything about protein purification must now be known. And even if it isn’t, and there
is something new to discover, how on earth could a CDT student make a contribution?
By thinking, that’s how. And thinking in the right way. Let’s suppose, for example, that the 2025 Nobel Prize for
Chemistry is given for a novel method for protein purification. Whatever that advance might be, one thing must be
true – that the new method will be a combination of pre-existing technologies, technologies that probably exist
today. That, after all, is exactly what Koestler’s Law implies. How, then, might this new combination be discovered?
One method is by luck – some scientist somewhere just happens to be in the right place at the right time, and
notices the combination. But another method is by deliberate search – by deconstructing everything we know about
existing methods, and asking “how might this be different?”. For sure, this is laborious and difficult – if it were easy,
everyone would be doing it. But something that is laborious and difficult is, at the same time, feasible and
tractable. And yes, there are many blind alleys to go down – but with wide knowledge and some imagination, and by
exploiting the human brain’s wonderful ability for pattern recognition in a context of ‘safety’, it’s totally, totally
possible.
So, on the afternoon of 8th September, the team had the opportunity to do just that. Pages 38 to 43 record the main
elements of the discussion, and, as will be seen, in just a few hours, two key themes emerged – the search for
alternatives to maltose binding protein, and the rearrangement and combining of conventional sequences of
chromatography. These, of course, are just fragments, glimpses of possibilities – which is how all new ideas begin.
Who knows where they might lead?
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Silver Bullet
Some key features of what we do now
The problem
Protein properties
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Charge.
Size.
Molecular weight.
Primary sequence.
Folding.
Orientation.
Stability.
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Affinity. (To what?)
Solubility.
Hydrophobicity.
Conductivity (electrogel).
Magnetic properties???
…
Strategies
 If the protein can be found, naturally, outside the cell, this
makes things easier.
 We need to exploit a property which distinguishes the target
protein from everything else…
 …which, in practice, might best take place in a sequence of steps…
 …each of which separates the target from successive classes of
contaminant…
 …so progressively purifying the target protein.
 At each stage, we need to assay the target protein…
 …so enabling the progress of the purification to be monitored…
 …and to assess how much of the target is lost at each stage.
 It is sometimes possible to modify the target protein, within the
cell, perhaps genetically, to influence a useful property, such as
a particular affinity (for example, His-tags), fluorescence, or
solubility, as achieved by the attachment of appropriate ligands.
Solubility…
…is influenced by…
Attachments
Surface charge
Media: salts, …
Attachments…
…for example, Maltose
Binding Protein (MBP)…
 …
 Must be reversible or detachable.
 Might affect tertiary structure.
 …
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How might this be different?
first draft 16th september
Proteins are synthesised within cells…
…and are located in the cytoplasm…
…or embedded in membranes…
…along with a large number of other proteins…
…and lots of other stuff too…
…so the protein purification problem is all about
how to create a homogeneous sample of the
protein we want…
 …with all other molecules (except the solvent)
absent…
 …without denaturing, or otherwise impairing the
action of, the target protein.




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38
Enhancing solubility
 What else, other than maltose, might bind suitably to MBP? For example…
 …lactose…
 …sucrose…
 …glucose…
 …ribose…
 …?
 What other solubilising agents might there be which are not sugars?
 What other natural solubilising proteins are there, like MBR, but different?
 The most general case is some from of molecular ‘bridge’, one ‘end’ of
which binds to a solubilising agent such as maltose, and the other binds
to the target protein, and can subsequently be cleaved.
 Suppose the gene were modified so that a second protein is expressed
along with the target protein. If this second protein has properties
which enable it to be purified relatively easily, might it ‘drag’ the target
protein along with it?
Reversibility and cleavage
 An ‘automatic’ assumption we inevitably make is that
anything attached to the target protein – such as
MBP – must later be detached, to return the target
protein to its original state. This makes sense, and
we all understand it…
 …but is it a necessary condition? Suppose we could
discover an attachment that makes it much easier,
say, to solubilise the target protein, and then
purify it. Suppose further that we can’t remove it –
but that we can verify that the ‘extra’ bit we’ve
added on to the target does not materially damage
the property we’re interested in. If this were to be
the case, then we have removed a constraint –
cleavage – from the overall process…
Some actions we could actually take…
 “Blast” the DNA sequence of MBP to find analogues.
 Search for some new ‘kick-out indicators’ – methods or signals (fluorescence?) that verify that the target protein
and the attached ligand have successfully been separated.
 Work on Increasing binding affinity, perhaps by using mutogenesis.
 Search for similar metabolisms/mechanisms.
 Search for alternatives to maltose, and see how easy they are to work with.
 Search for alternatives to MBP, and see how easy they are to separate from the protein (if this is in fact necessary).
39
first draft 16th september
How might MBP be different?
Silver Bullet
Chromatography
How might chromatography be different?
This page captures the general essence of chromatography-as-we-do-it-today
Initial
protein
mix
Affinity
Operational options
Purified
mixture 1
Cation
exchange
Anion
exchange
or HIC
Purified
mixture 3
Column options
Analytics
 Packed bed
 single ligand
 multicore, with two ligands.
 Monoliths.
 Expanded bed.
 Membrane chromatography.
 HPLC
 Reverse phase
 SEC
 IEX
 AFF
 HIC.
 Two-dimensional gels.
 Biosensors (?).
 Capillary electrophoresis.
 …
 Batch.
 Continuous.
Silver Bullet
Purified
mixture 2
40
first draft 16th september
Of all the techniques used for protein purification – techniques such as precipitation, ultracentrifugation, electrophoresis,
crystallisation and so on – chromatography is probably the most widespread and versatile.
Initial
protein
mix
Mixed mode
Purified
mixture 1
Initial
protein
mix
Cation or anion
exchange
Purified
mixture 1
Initial
protein
mix
Mixed mode
Purified
mixture 1
Initial
protein
mix
Liquid-liquid
exchange
Purified
mixture 1
41
Cation or anion
exchange
Purified
mixture 2
Mixed mode
Purified
mixture 2
Liquid-liquid
exchange
Mixed mode
Purified
mixture 2
Purified
mixture 2
first draft 16th september
Potential new patterns of chromatography…
Silver Bullet
Silver Bullet
42
first draft 16th september
 Current methods of protein purification rely on a sequence of individual steps, each utilising a particular
property – so, for example, an initial stage of ultracentrifugation separates proteins according to molecular
weight, and a subsequent stage of, say, chromatography can then take any weight fraction and purify
further according to, for example, affinity.
first draft 16th september
A BIG IDEA – two simultaneous fields
 What would happen if two properties were exploited simultaneously? So, for example, imagine an
ultracentrifuge which, instead of being a set of test-tubes, arranged like spokes on a wheel, each containing
an aqueous sample, were a circular sheet of gel, arranged in a plane perpendicular to the axis of rotation,
and with the axis of rotation at the centre (rather like how a pleated skirt flares out horizontally as a
dancer rotates). Suppose further that, say, an electric field were applied, in the plane of the gel but
tangentially.
Any molecule in the matrix of the gel would then be subject to two orthogonal fields, and
the corresponding forces. As the gel rotates, the centrifugal field ‘flings’ the molecule radially away from the
axis of rotation,
towards the outer periphery, with heavier molecules moving faster than light ones.
Simultaneously, the
tangential electric field would cause migration perpendicular to the radius,
according to charge.
 That’s only a ‘back-of-the-envelope’ description of an initial concept… but perhaps it’s the ‘germ’ of a
potentially new idea – to use two effects, simultaneously, perhaps making protein purification faster, and
even more efficient.
 And, in accordance with Koestler’s Law, there’s nothing new here. The idea of having two fields – such as
orthogonal electric and magnetic fields - has been used for decades in particle physics, and also forms the
basis, for example, of velocity selection in mass spectrometry. Mass spectrometry is, as we all know, used
primarily to measure mass, as achieved by separating a mixture of ionised particles; looked at in a rather
different way, this separation of the components of an initial mixture is precisely what protein purification
is all about. And let’s note here that John Fenn and Koichi Tanaka shared the 2002 Nobel Prize for
Chemistry for their development of techniques of mass spectrometry for biological macromolecules…
43
Silver Bullet
“In a collaboration, the benefits must be mutual.”
“A collaborator is someone who needs your expertise in return.”
“The right collaborator is, above all, willing, and has the right resources –
especially time - available. They need to be able to communicate with you,
and for that communication to be mutual.
“Collaborations are built on trust, and real trust is hard to build. How can
you start? Here’s an idea: during our time as CDT students, we should have a
number of short (say, 4 week?) mini-projects which we do collaboratively in
pairs or small teams. That will give us all a series of experiences working
together – experiences long enough to build trust, but short enough that not
too much pain is endured if things don’t work so well. Certainly, an issue-tomanage is the identification and organisation of suitable projects. But surely
that can be solved…”
Silver Bullet
44
first draft 16th september
“You can’t expect others to help you if you
don’t help others.”