Protein isolation is one of the oldest ‘biotechnologies’, but the demands from proteomics for the purification of potentially vast numbers of proteins is driving new developments in long-established techniques. “Researchers working in genomics are looking for the next step to fully understand the results of their sequence analysis — the proteins that the genome expresses — in the context of systems biology,” says Anke Cassing, associate director for corporate strategy at Qiagen in Hilden, Germany. “The delicate interplay of proteins is, of course, also of extreme interest to pharmaceutical companies, which are always on the lookout for new drug targets.”

There is increasing demand from researchers producing biopharmaceuticals — antibodies and proteins used as drugs. “There's a driving force towards protein purification, separation and analysis,” says Carsten Buhlmann, product manager at Agilent, based in Palo Alto, California. “Especially for the biopharmaceuticals, there's a high demand for purity of these proteins that are used for drugs and have to get through all the regulations.”

Anke Cassing: systems biology is the next step. Credit: QIAGEN

For virtually all applications, researchers need to maintain a protein's biological activity, which can rule out some purification processes. Proteins can be fragile and easily denatured, and many of the most important are insoluble in the most common media.

“If you look at the average protein, it's quite complex, it's a buzzing, vibrating molecule — it's not a fixed structure,” says Allan Simpson, vice-president for product development at the protein separations division of GE Healthcare Biosciences in Uppsala, Sweden. “They're very hard to handle, they're difficult to purify, they can aggregate easily — these are very hard things to manipulate.”

Proteomics workhorse

With proteins taking centre stage in many laboratories, equipment developers are rolling out a new generation of automated systems to take the grind out of separating and purifying proteins of interest. Chromatographic separation is one of the basic protein-purification techniques and one platform that is emerging as a workhorse of the large proteomics lab is ÄKTAxpress. Made by GE Healthcare, this is a dedicated high-throughput multistep chromatography system for purifying histidine (His)- and glutathione-S-transferase (GST)-tagged recombinant proteins.

GE began developing the platform in the late 1990s after realizing that there were not enough trained chromatographers to produce proteins in the quantities and varieties demanded by post-genomic researchers. “We decided to see if we could automate a system that would be better than the current technologies at solving that problem,” Simpson says. “Instead of taking a robot and automating the current system, we set out to develop a new one. A robot represents nothing more than a mechanical technician — it reproduces all the successes but also all the errors, so you don't move forward in your science.”

The company produced a high-throughput system that needs no specialized knowledge to operate and can be programmed to carry out up to four common purification steps starting with affinity purification. “The skill of chromatography is sitting within the system,” says Simpson. The software is written as a series of wizards, each representing a single step. GE will shortly be launching a software package for the purification of monoclonal antibodies.

The basic four-module set-up can purify up to 2,500 proteins a year, each module producing up to 50 mg of protein per run. A twin-pack version is aimed at smaller labs wanting to purify up to 1,000 proteins a year.

Scaling-up: ÄKTAxpress at Monash University's protein purification facility.

The new protein-purification facility at Monash University in Melbourne, Australia, is deploying a 12-module ÄKTAxpress set-up for its ambitious development programme. “I'm a structural biologist, so my interest is in producing large amounts of recombinant protein for structural and functional studies,” says James Whisstock, scientific director of the facility. “What's really important is that the cost of equipment is within reach of a normal university laboratory set-up. There are some very big structural biology institutes with between US$50 million and $100 million's worth of industrial-scale protein preparation equipment. From our point of view that's not achievable, but we're bringing in this technology, which is going to make a huge difference to our research.”

The ability to deal with many more proteins simultaneously will allow the lab to approach problems differently. “If you have a very challenging protein target and want to try 50 different constructs, at the moment it's really not feasible to do that manually one after the other,” Whisstock points out. “Now, you can try the same molecule from 50 different species. With parallel advances in expression technology, the whole process is simplified and really streamlined, and provides the capacity to perform that experiment. You're trying so many different things simultaneously, you're likely to get a result.”

Automating innovation

Several companies are developing equipment and product ranges that can be used at different points in the proteomics pipeline, from raw cell extracts to mass spectrometry and beyond. Beckman Coulter in Fullerton, California, is rolling out its ProteomeLab family to help with everything from initial purification of cell extracts, through protein fractionation and characterization, to the ultimate steps of disease diagnosis.

“We try to link technologies together to simplify the job for what takes place at the end, which is typically mass spectrometry,” says John Hobbs, group product manager for ProteomeLab. “To get to that point, a lot of people have realized that it's garbage in, garbage out. If you put crap into a mass spectrometer, the results you get out will be the same.”

The ProteomeLab PF 2D Protein Fractionation System automates two-dimensional chromatographic fractionation, resolving proteins by isoelectric point and hydrophobicity. The emphasis is on standardizing the protocols and techniques used by researchers. “Biologists want to be able to look at their results and see if they relate to someone else's,” Hobbs notes. “As well as providing the instrument, we provide the methodology and buffers, but we do specify you have to use that method to get the full support. It's a little bit different from the normal research instrument approach, but we thought there was a need for that and it seems to be accepted.”

Beckman is also currently commercializing an innovative system of protein partitioning using affinity fractionation to decrease the unwanted complexity of protein mixtures before analysis. The aim is to remove not just very abundant proteins, for example serum albumin from blood plasma, but also other proteins that are already well characterized. The firm claims that up to 95% of the proteins in a cell lysate can be removed before the full fractionation stage with less risk than other purification procedures of losing the proteins you're interested in. “There's a growing interest in what might be going away with these large-abundance proteins — albumin is a binding protein, and possibly some interesting proteins go with it,” Hobbs notes.

Several big equipment producers have teamed up with smaller specialist firms to include cutting-edge reagents or media in application kits for their automated systems. Tecan in Männedorf, Switzerland, recently signed a licensing agreement to deploy the paramagnetic beads developed by Dynal Biotech in Oslo, Norway, on its Freedom EVO liquid-handling platform (see ‘Attracting attention’, page 796). The Robopop protein purification kits from Novagen in Madison, Wisconsin, can also be used on Tecan's workstation and on the MultiPROBE liquid-handling workstation from PerkinElmer in Boston, Massachusetts.

The LabChip90 from Caliper Life Sciences.

Caliper Life Sciences in Hopkinton, Massachusetts, has integrated into its Sciclone ALH3000 liquid-handling workstation a new column technology developed by PhyNexus based in San Jose, California (see ‘Small-scale separation’, page 795). The combination allows researchers to purify and enrich small quantities of up to 96 engineered proteins in as little as 15 minutes.

The market for protein purification systems has changed in the past six months, notes Mark Roskey, vice-president of marketing at Caliper, with more groups getting involved in larger-scale protein purification. “It's not at the industrial scale, but regular pharma and biotech R&D people are now trying to purify proteins in a more parallel situation. A lot of this stems from having all the genes and working with huge numbers of them to develop new drugs,” he says.

Analysis and optimization

To maintain the benefits of high-throughput separation and purification, the proteins of interest must be able to pass smoothly into the next stage of the process. “Once you've got a relatively pure protein you need to determine whether it is pure, so there's issues with analysis as well,” says Roskey. A common analysis method is SDS-polyacrylamide gel electrophoresis “but we feel that that is a bottleneck”, Roskey adds.

Although the established protocols of macroscale gel electrophoresis are being successfully automated (see ‘Automation in two dimensions’, below), many users are turning instead to microfluidic and lab-on-a-chip solutions. In January, Caliper launched the Protein Express Assay for its LabChip 90 automated electrophoresis system.

“It's a microfluidic replacement for SDS-PAGE that automates the whole process,” Roskey says. “Rather than putting samples on a gel, you get them off a multiwell plate, and it does integrated separation, detection and analysis. It can do an individual protein in around 30 seconds, so you can do 96 proteins in an hour with the same quality as SDS — in fact better, because the data are digital.”

Automated electrophoresis using chips developed with Caliper is the basis of two new systems — the bench-top Experion from Bio-Rad in Hercules, California, which analyses a single Pro260 chip carrying a maximum of 10 samples in 30 minutes for the medium-scale user, and the ultra-high-throughput 5100 Automated Lab-on-a-Chip system from Agilent in Palo Alto, California. The latter is aimed at pharmaceutical companies and other laboratories needing to separate, purify and analyse thousands of proteins a day. The fully automated 5100ALP using the Protein 200 Plus LabChip kit can take up to twelve 96- or 384-well plates for overnight analysis, with each chip capable of up to 6,000 sample runs. “The real innovation with the 5100 is that you have a complete unattended solution ending up with digital data,” says Carsten Buhlmann, product manager for Agilent's microfluidics group.

Large-scale protein production needs equally high-throughput analysis. Credit: ASTRAZENECA

The 5100ALP has been deployed at the centralized protein-production facility for AstraZeneca in Alderley Park, UK. “We're developing a high-throughput protein-production platform and wanted a quantitative and qualitative data-analysis method for that process,” says Paul Hawtin, senior research chemist at AstraZeneca's UK protein group. “Previously we used SDS-PAGE gels and although they get you the information you need, they're very cumbersome, especially when you're using the numbers that we're using.”

With high-throughput protein production, an integrated analysis capability is invaluable. “We were in the situation where we could do high-throughput molecular biology, high-throughput expression and also high-throughput production — once you've gone through that process and you're testing a number of variables, the numbers you've got coming out the back end are incredibly large,” Hawtin notes. The AstraZeneca team also uses the ÄKTAxpress to follow up with larger-scale protein production.

Kits and columns

Researchers who don't need automation can choose from an increasing selection of specialized purification and fractionation kits. Kits for affinity purification of recombinant proteins tagged with His6, GST, streptactin-binding Strep-tag, streptavidin-binding peptide and other tags abound, and many incorporate magnetic bead technology.

On the protein fractionation and preparation front, in January Qiagen launched a new line of protein fractionation kits under the Qproteome brand. The range features kits, including reagents, buffers and columns, for such common tasks as phosphoprotein purification, glycoprotein fractionation and albumin depletion.

“The simplicity of the kits and procedures offers a gentle introduction to the world of protein science for molecular biologists who might be wary of having to learn new techniques or understand complex technologies,” says Cassing.

Many protein purification kits based on filtration are available in spin-column format, allowing faster processing. Vivascience, a subsidiary of Sartorius, based in Hanover, Germany, has developed a range of specialized spin-column purification kits, based on the firm's membrane matrix of stabilized regenerated cellulose. Because of the porous structure, the surface available for contact and binding is about 100 times that of the same volume of traditional bead-based resins, allowing parallel separation of proteins with high yields in less than 20 minutes.

The new ProteoSpin line of spin column kits for protein clean up from Norgen Biotek in St Catharines, Ontario, is based on a patent-pending technology using modified silicon carbide (SiC) as the matrix rather than the usual silica (SiO2). SiC has all the benefits of silica resin and more, says Yousef Haj-Ahmad, president and chief executive of Norgen Biotek. The hydrophobic and hydrophilic surfaces of SiC can be exploited directly rather than having to chemically add active sites, as with a silica matrix. “The lack of porosity is an advantage because it enables the purification of a wide size range of proteins,” Haj-Ahmad notes. “With silica-based resins, even if they are ion-exchange type, one finds size restrictions for proteins because of the micropores.” Also, the unique way that SiC's surface charges allow it to function as an ion exchanger means that salts are not needed to elute proteins in most cases. The first ProteoSpin kits are focused on protein preparation for downstream applications such as mass spectrometry.

The next challenge

Many of the large equipment providers are now concentrating on streamlining and speeding up the overall protein-processing workflow, from improving initial sample clarification through to a smoother transition to mass spectrometry, microarray technology or X-ray crystallography. Informatics at the back end also remains a challenge, in terms of analysing the results of large-scale analysis and feeding them back into the production process.

Allan Simpson: membrane proteins will be “the pot of gold”. Credit: GE HEALTHCARE

More sophisticated successors to the His6 and GST tags commonly used in protein purification are also being sought. “Most people are still using the classic protein purification tags that have been around for many years,” says Roskey. “There probably need to be some new advances in that area, better ways to grab hold of proteins as you express them. That's something that a lot of people are working on.”

But perhaps the biggest challenge is in applying the skills learned with soluble proteins to membrane proteins. “There's not a soluble protein that we can't purify for you,” says Simpson. “But once you get to membrane proteins, which are particularly interesting for the pharma industry, they're a nightmare. They're probably key receptors for most drugs but they're designed to be insoluble in physiological structures. How to address membrane proteins is one of the critical bottlenecks. Everyone's putting so much effort into it that it will be solved, and whoever solves it will really hit the pot of gold because it will change the face of our industry and the way medicines are designed.”