Protein Crystallization Hits

Some of you may know that Emerald BioStructures, as part of the Seattle Structural Genomics Center for Infectious Disease (SSGCID) has contributed to submitting more than 444 protein structures to the PDB in the past 4 years. That's quite an achievement and my congratulations go out to the project teams that are behind these structures, most of them determined via X-ray crystallography. Some of this output, including methods used to achieve this level of productivity, are described in the  September 2011 issue of Acta Cryst F.

One of the protein production methods that has been key for several of my own 2011 protein crystallization projects: lysis scouting with the Protein Maker instrument (described in this open access article "The Protein Maker: an automated system for high-throughput parallel purification". 

Smith, E., Begley, D., Anderson, V., Raymond, A., Haffner, T., Robinson, J., Edwards, T., Duncan, N., Gerdts, C., Mixon, M., Nollert, P., Staker, B., & Stewart, L. (2011). The Protein Maker: an automated system for high-throughput parallel purification Acta Crystallographica Section F Structural Biology and Crystallization Communications, 67 (9), 1015-1021 DOI: 10.1107/S1744309111028776

What is lysis scouting?

Stated simply, lysis scouting combines the testing of a set of cell-lysis buffer conditions with IMAC (ion metal affinity chromatography) . This is done to increase the yield of proteins that appear partially soluble or insoluble under standard lysis buffer conditions.  This procedure results in a clear path forward for scaled-up production of purified protein samples for protein crystallization trials.

How is lysis scouting done?

A single batch of protein expressing E.coli cells is split into 12 pools and lysed by sonication in 12 different buffer conditions. The paper shows as an example P450 51 A1 (CYP51A1) with a 6xHis-Smt tag. This is the outline of the lysis scouting protocol:

1. Prepare 12 aliquots, each corresponding to 3 g of wet cell paste
2. Resuspend in 30 mL lysis buffer (one out of an array of 12) - see table below.

Cell lysis buffers for testing lysis conditions of recombinantly expressed fungal cytochrome P450

3. Sonicate to lyse and spin to remove cell debris

4. Clarify lysates and load on 12 x 1 mL Ni-affinity matrix column

5. Wash, elute and analyze fractions

SDS-PAGE showing that buffers 1C and 1D extract much more of the target protein CYP51A1 (red boxes). L(load), W(wash) and E(elution) fractions are shown next to MW standards.

While well expressed, CYP51A seemed insoluble using standard cell-lysis methods. The lysis-scouting procedure yielded a buffer system with a detergent (CHAPS or octyl glucoside)  in the presence of high salt concentrations (500 mM NaCl).

The utility of the Protein Maker instrument in this process is the short time it takes to run a lysis scouting experiment. Total run time is approximately 1.5 hours (excluding sample analysis). I.e. many proteins can be tested for optimal lysis conditions in a single day - and since the instrument carries out the experiment for you and in parallel, there is plenty of time to strategize the next steps of mg-scale production of the protein sample for crystallization. 

There are many protein structures that we have produced in 2011 that would not exist without Protein Maker supported lysis optimization.

A true work horse.

Peter

Looking beyond your typical day-to-day work and finding out what ‘the rest of the world’ is doing can be a lot of fun. This is one of the reasons I’m enjoying myself so much at the PepTalk 2011 meeting that is currently taking place in San Diego. As I’m learning about the unique challenges faced in protein drug formulations I am quite surprised at the arsenal of analytical techniques that are available to investigate the ‘health’ of a protein prep.

With respect to protein crystallization there has been one presentation by a colleague of Bhami Shenoy from Althea that I found rather inspiring.

The purpose of biologics formulation is to provide a high concentration of pure, stable and functional protein sample in a form that can be kept for an extended period of time, for instance 2 years at room temperature, while showing little decay (no chemical decomposition, aggregation or particle formation). Apparently several protein microcrystaline suspensions are in late stage clinical trials and are an attractive dosage form for several reasons: high protein concentration (>100 mg/ml) solution with low viscosity (patients like their injections to be quick and painless), high stability and slow release of the protein into the bloodstream (lack of burst).

The part of the talk I enjoyed most was a comparison between the requirements of X-ray protein crystallography with that of biologics formulation. Here's a short summary:

Overall, the different requirements are driven by the need for low process costs in biologics formulation. After all, short timelines reduce cost.  Curiously, the choice of precipitation is rather limited as compared to what we do for crystal growth for X-ray diffraction experiments, since the crystal suspensions are to be injected into people (better not use that cacodylate buffer system that X-ray crystallographers appear to like so much). The toolset consists of a variety of pH values, a few salts, dicarboxylic acids, sugars, amino acids and detergents. No ammonium sulfate, no polyethylene glycol, no MPD. This makes the identification of productive crystallization conditions rather difficult.

Original data on Infliximab and long acting human growth hormone was presented, showing lowered viscosity (and what was called ‘syringibility’), low/no toxicity or injection site reactions, improved stability and extended release profile while retaining activity and efficacy. I was also impressed by the fact that protein therapeutics crystallizations at production scale are typically carried out in batches of several hundred liters, producing kilo gram amounts of protein crystalline material. I’ll think about that next time I’m setting up a 0.5 + 0.5 microliter vapor diffusion protein crystallization experiment.

Greetings from sunny San Diego,

Peter

Starter for biologics crystallization:

Jen A, & Merkle HP (2001). Diamonds in the rough: protein crystals from a formulation perspective. Pharmaceutical research, 18 (11), 1483-8 PMID: 11758753

When you think about the 'front end' of a protein crystallization project, E.coli is front and center. These bacteria are used to create designed vector constructs and are the standard vehicle for heterologous protein over expression. However, one could also chemically synthesize the protein from scratch - a notorious example is Lysozyme, the 1.04 Å X-ray structure of which has been obtained from crystals grown from a sample that had been obtained by total chemical synthesis. An earlier example for such a path is the structure of the anti-HIV protein AOP-RANTES. 

Chemical synthesis is a tour de force and not a practicable path for most of us. Now though, protein synthesis without involving a chemical fume hood nor culturing E.coli, or any living organisms for that matter, can be done in a standard lab. There are several reports of crystallographic structures that were produced from crystals that had been grown from protein material obtained in cell-free systems. The protein producing machinery is still of biological origin, though. So technically, living organisms are involved - but the timing of their cultivation and the protein production is uncoupled. This circumvents the need to cultivate cells in your own lab and could be done away with altogether when the DNA is of synthetic origin as well.

Here are two X-ray crystallographic protein structure reports that are based on crystals grown from in-vitro expressed protein:

The case for retiring E.coli and utilizing cell-free systems can be made especially for those proteins that are expressed at low yield or that are toxic to E.coli or any other cells (i.e. DNA modifying enzymes) or when specific labels need to be introduced into the protein. Direct access to the protein synthesis machinery is unique and allows tackling difficult targets, such as membrane proteins. A recent summary of such ongoing research is to be published here:

And of course, one of the reasons I mention this synthetic biology route has to do with the fact that we offer via Emerald BioSystems the wheat-germ based protein expression system. Our partner in Japan, Cell Free Sciences has developed reagents and a sophisticated robot that enables researchers to produce milligram amounts of protein. The robot is called Protemist DTII. All you need to do is load the instrument with target-DNA and reagents, klick a button on the screen and walk away. When you're back after one and a half days the instrument has produced (via transcription, translation and affinity purification) your purified target protein. Pretty convenient, isn't it? The instrument that may be most interesting to protein crystallographers though, is the new Protemist XE, shown below. Its capacity is designed to produce tens of milligrams of protein within a one or two day campaign. 

No living cells involved: ten milligram of GFP produced with the Protemist XE using the wheat germ cell-free expression system.

Drop us a note (sales@emeraldbiosystems.com) if you're interested in more information about this protein production system (and tell Frank that Peter sent you :)

Regards,

Peter

P.S. I just saw this comprehensive review article in Nature Biotech, covering the subject of cell-free protein synthesis for functional and structural analysis of membrane proteins:

Junge F, Haberstock S, Roos C, Stefer S, Proverbio D, Dötsch V, Bernhard F.
Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins.
N Biotechnol. 2010 Jul 15. [Epub ahead of print]

Structure, the magazine, sent me an email the other day with "the most requested full text articles your colleagues and competitors are currently downloading from Structure.". OK - let's see what's there for us protein crystallizers:

1. Conformation Dependence of Backbone Geometry in Proteins
2. Structure of the Human Dicer-TRBP Complex by Electron Microscopy
3. The Nuclear Pore Complex Has Entered the Atomic Age • Review article
4. Discovery Through the Computational Microscope • Review article
5. Structure of PAS-Linked Histidine Kinase and the Response Regulator Complex
6. Structure of IL-33 and Its Interaction with the ST2 and IL-1RAcP Receptors-Insight into Heterotrimeric IL-1 Signaling Complexes
7. Insights into How Nucleotide-Binding Domains Power ABC Transport
8. Structural Insights on the Mycobacterium tuberculosis Proteasomal ATPase Mpa
9. Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins
10. Secondary Structure of Huntingtin Amino-Terminal Region
11. Structure and Signaling Mechanism of Per-ARNT-Sim Domains • Review article
12. Intrinsic Domain and Loop Dynamics Commensurate with Catalytic Turnover in an Induced-Fit Enzyme
13. Quantitative Determination of the Conformational Properties of Partially Folded and Intrinsically Disordered Proteins Using NMR Dipolar Couplings • Review article
14. Structural Plasticity of Eph Receptor A4 Facilitates Cross-Class Ephrin Signaling
15. Structural Dynamics of Light-Driven Proton Pumps
16. Subunit Architecture of Multiprotein Assemblies Determined Using Restraints from Gas-Phase Measurements
17. Toward Structural Elucidation of the @c-Secretase Complex • Review article
18. Structural Basis for DNase Activity of a Conserved Protein Implicated in CRISPR-Mediated Genome Defense
19. Structural Basis of Novel Interactions Between the Small-GTPase and GDI-like Domains in Prokaryotic FeoB Iron Transporter
20. Structures and mechanisms of glycosyl hydrolases
21. Binding of Small-Molecule Ligands to Proteins:''What You See''Is Not Always''What You Get'' • Review article
22. Structural Basis for Calcium Sensing by GCaMP2
23. Structural Basis for Binding of Hypoxia-Inducible Factor to the Oxygen-Sensing Prolyl Hydroxylases
24. The Origin of Allosteric Functional Modulation: Multiple Pre-existing Pathways • Review article
25. A Specific Cholesterol Binding Site Is Established by the 2.8 A Structure of the Human @b"2-Adrenergic Receptor

I am rather surprised by the high number of membrane protein-related papers. Out of these 25 hottest STRUCTURE articles I'd say that #9 looks like the most interesting one:

T. Kawate & E. Gouaux (2006)

Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins

Volume 14 (4), 673-681

And this is of course Eric Gouaux & Toshimitsu Kawate's clever paper on analyzing the monodispersity and stability of protein-detergent complexes: 

Schematic pre-crystalline screening for membrane protein crystallization.

Described is a "rapid and efficient precrystallization screening strategy in which the target protein is covalently fused to green fluorescent protein (GFP) and the resulting unpurified protein is analyzed by fluorescence-detection size-exclusion chromatography (FSEC). This strategy requires only nanogram quantities of unpurified protein and allows one to evaluate localization and expression level, the degree of monodispersity, and the approximate molecular mass." This helps leapfrogging the otherwise tedious step purification optimization. And it helps to reduce "...unproductive large-scale protein expression and purification..." efforts.

Well done!

Peter

X-ray crystallography is only one of several disciplines requiring protein crystals as essential materials. Some of us X-ray centric structure biologists may actually be surprised that there's a use for protein crystals for anything else besides X-ray diffraction. Here's my run-down:

1. Neutron diffraction
You're right - this is a lame one, protein structure determination with neutrons instead of X-rays. To diffract a neutron beam you need protein crystals. Massive ones. Think milli meters, not micro meters. But you'll see hydrogen atoms and their 'gymnastics'. Here's Dean Myles arguing for a bright future of neutron diffraction.

2. Solid State Nuclear Magnetic Resonance
Protein crystals sizes under 1 um? No problem. The good news with ssNMR: nanocrystalline material is fine.

Martin RW, Zilm KW.
Preparation of protein nanocrystals and their characterization by solid state NMR.
J Magn Reson. 2003 Nov;165(1):162-74.

3. Protein Purification
Crystallization is a way to purify and enrich your target protein, remember?
Seriously, I predict that we'll be seeing a renaissance of protein crystallization as a purification method for bulk material processing.

Russell A. Judge, Michael R. Johns, Edward T. White
Protein purification by bulk crystallization: The recovery of ovalbumin
Biotechnology and Bioengineering, Volume 48 Issue 4, Pages 316 - 323 (2004)

Why? Columns are expensive, crystallization is cheap.

4. Protein Formulations
Many small molecule drugs are delivered as carefully formulated crystalline powders. The same can be done for protein therapeutics. See:

Alexey L. Margolin et al.
Stabilized protein crystals, formulations comprising them and methods of making them
US Patent 7,351,798 B2

This is a patent claiming dried protein crystals formulation as a means to provide slow release for biomedical delivery applications such as vaccines and modern biologics protein drugs.

And this may be an interesting primer if you're interested in the use of protein crystals for protein formulation design.

Anna J., Merkle, H.P.
Diamonds in the Rough: Protein Crystals from a Formulation Perspective (great title!)
Journal Pharmaceutical Research
Volume 18, Number 11 (2001)

Plenty of job opportunities out there for protein crystallizers!

Peter