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