Proteomics aims to identify the cellular functions of all proteins encoded by the genome of an organism. Protein structure determination, proteome-wide functional screens, and the identification of protein interactions are just a few of the proteomics applications requiring thousands of purified proteins as a starting point. Researchers now have many reagents and off-the-shelf kits for the isolation of proteins to 90% purity. The challenge facing researchers is to adapt these methods to purify hundreds of different proteins in parallel using robotic systems. Several years ago a small number of academic institutions and biotechnology companies responded to the challenge by developing pipelines for high-throughput protein purification (see “Proteomics at Harvard”). Some of the reagents and instruments required for these methods are making their way to the marketplace.

HT purification

One widely used method for purifying hundreds of proteins in parallel involves their expression in a heterologous system. Once a target protein is identified, the corresponding complementary DNA is cloned into an expression vector for producing the protein in Escherichia coli or another organism. The purification of the expressed recombinant protein from bacteria typically involves cell lysis, incubation of the lysate with an affinity resin, washes and elution. Additional purification steps, such as ion-exchange and size-exclusion chromatography, are required for protein crystallography (see “Protein purification for structural proteomics”).

One bottleneck in high-throughput protein production is the expression of enough properly folded protein in bacterial cells. Proteins expressed in E. coli can accumulate as inactive, misfolded aggregates called inclusion bodies. Proteins in this form can be purified under denaturing conditions, but need to be refolded into their native conformations. A number of groups have developed procedures for expressing proteins in insect cells or cell-free extracts to get around some of these problems (see “Cell or cell-free?”).

Splitter: Pall's AcroPrep 96 Filter Plate. Credit: PALL CORPORATION

Each protein is expressed in different amounts and has different properties. Thus, to be able to apply the same purification protocol across the range of proteins, researchers have engineered proteins with generic tags that will bind to an affinity ligand. Widely used tags include a small peptide of six histidine residues (6xHis), a calmodulin-binding peptide, the streptavidin friendly biotin, the cellulose-binding tag, the maltose-binding protein (NusA), and glutathione S-transferase (GST). Vectors designed for expressing tagged proteins can be purchased from several manufacturers including Invitrogen (Carlsbad, California), Novagen (Madison, Wisconsin), Roche Applied Science (Indianapolis, Indiana) and others.

Some companies are now developing high-throughput methods for purifying proteins that are not tagged. “Typically most scientists work with tagged proteins through the discovery process. However, it is possible to lose some information because of tag interference in the biological assays. Removing the tag means removing some of the question marks,” says Lisa Bradbury, director of R&D for proteomics and cell therapy at Pall Corporation of East Hills, New York.

Purification tool bag

High-capacity purification: Teledyne Isoc's BioOptix 10. Credit: TELEDYNE ISCO

Several companies sell reagents for every step of the purification process — from cell lysis, to affinity purification resins, to desalting and concentrating reagents — adapted to high-throughput protocols that use standard 96-well plates. “We were recognized for our protein affinity-purification line. Now we are configuring it into a format that is automation-friendly,” says Craig Smith, vice-president of R&D at Pierce Biotechnology of Rockford, Illinois (part of Fisher Scientific). Among the company's offerings are the SwellGel Discs, pellets of dehydrated support that can be distributed into filter plates. When the protein is added, the disc rehydrates to an affinity gel that binds tagged proteins. “We have taken out variability by having a dried pellet,” says Smith. More recently, the company's Zeba Micro Desalt Spin Columns were configured to 96 well-plate format for desalting many small samples in parallel.

Pall Corporation also sells reagents for each step of the purification process. The AcroPrep 96 Filter Plate can be used for any type of chromatography, including immobilized metal affinity chromatography, in a multiwell platform. The format facilitates the optimization of various purification parameters, including the choice of metal ion and resin, the sample-to-resin ratio, and the elution conditions. Other reagents adapted to a high-throughput environment include the Mustang 96-well ion-exchange plates and Nanosep Centrifugal Devices.

QIAGEN, based in Hilden, Germay, offers the Superflow 96 BioRobot, a medium-scale purification kit for 6xHis-tagged proteins. “You load your pellets onto a robot, press a button, and you will have product in two hours,” says Frank Schäfer, associate director for R&D protein expression/proteomics. According to the company, purification can be done in denaturing or native conditions and yields 4 mg of protein for each of 96 wells. “It is one system and one process for every sample,” says Schäfer. QIAGEN sells a similar system that runs up to 24 samples in parallel to purify up to 30 mg of protein for large-scale applications.

Novagen's host of reagents for high-throughput protein purification starts with the expression step. The Overnight Express Autoinduction Systems allow fully automated, parallel protein expression from E. coli cultures, and achieve high cell densities without monitoring growth or induction by the addition of isopropyl-beta-D-thiogalactopyranoside. “The medium contains a blend of carbon sources optimized for tightly regulated, uninduced growth and automatic induction of protein expression,” says Anthony Grabski, R&D group leader for protein purification. The RoboPop Purification Kits contain reagents for the extraction and purification of His or GST fusion proteins by magnetic or filtration-based affinity purification protocols, all in a 96-well format. The kits have been validated on both PerkinElmer and Tecan robotic liquid handlers.

Other providers of reagents for high-throughput protein purification include GE Healthcare of Little Chalfont, UK, with its Tricorn High Performance Columns designed for high-resolution purification of proteins, peptides and other biomolecules. Sartorius of Göttingen, Germany, introduced the Vivapure 8-to-96 well cobalt chelate kit for the simultaneous purification of multiple His-fusion proteins by affinity chromatography. And St Louis-based Sigma-Aldrich's HIS-Select iLAP Plates are plates coated with cell-lysis reagents and a HIS-Select nickel chelate matrix allowing for cell lysis, protein capture, and assay of a His-fusion protein in a single well.

As well as purification kits, several companies offer products to help screen the outcome of each step in the purification pipeline. Novagen's RoboPop Solubility Screening Kit contains a filtration plate that retains insoluble inclusion bodies while allowing soluble proteins to be collected for rapid quantitation and analysis. “You can screen expression conditions for soluble protein before you proceed to purification,” says Grabski. In January, the company released the iFold Protein Refolding System 1 to screen different refolding conditions in parallel. The system includes inclusion-body purification reagents and 92 different refolding buffers. “Structural proteomics groups have harvested most of the low-hanging fruits, so now they will have to focus on difficult proteins that are insolubly expressed,” says Grabski. Another product for monitoring the purification procedure is the Protein 200-HT2 assay of Agilent Technologies (Palo Alto, California), which allows the identification, sizing and quantification of proteins from 14 kD to 200 kD in size. “It allows researchers to replace SDS–PAGE procedures in the lab,” says product manager Carsten Buhlmann. Finally, some reagents and instruments have been designed to help researchers hone in, for example, on medically relevant targets, which can then be purified and studied (see “Target identification”).

Getting automated

Mix and match: PerkinElmer's JANUS workstation. Credit: PERKINELMER

The available reagents and kits can be used to purify several hundred proteins in a 96-well format at considerable speed. However, the tedious and error-prone nature of manually performed high-throughput operations calls for automation of the process. “When a robot is doing the pipetting there are no mistakes, so you can have greater consistency and reproducibility,” says David Daniels, applications marketing manager for Beckman Coulter in Fullerton, California. Liquid handlers perform all the steps of protein purification from loading cell cultures to obtaining a protein in solution, in under four hours.

Two leading liquid handlers are Beckman Coulter's Biomek 3000 and Biomek FX instruments, with plate-deck configurations that can handle 10 and 18 plate positions, respectively. The Biomek 3000 will process two 96-well plates in 2–3 hours; the FX twice as many.

Promega in Madison, Wisconsin, automated high-throughput protein purification using its MagneHis Protein Purification System and the Biomek FX instruments. The MagneHis reagent contains a cell-lysis solution that allows resuspension and lysis of bacterial cell pellets without sonication and centrifugation. Magnetic pre-charged nickel particles are then used to isolate His-tagged proteins from the cell lysate. The MagneHis particles bound to the target proteins are captured on a MagnaBot 96 Magnetic Separation device — a plate that can be hooked up to the Biomek instruments.

In January, PerkinElmer (Boston, Massachusetts) launched its modular and scalable JANUS Automated Workstation. “Many units can be configured at the time of sale,” says Nance Hall, business unit leader for automation and liquid handling. “With JANUS, you can scale and change the system not only when you buy, but also in the future.” Users can also program the selection of numerous dispense heads and formats. “It is as though you go into a kitchen and you are asked if you need a cup or a teaspoon,” explains Hall. “In the kitchen you will use both.” Likewise, the JANUS allows users to choose different tools automatically depending on the volume range and microplate densities.

Another popular choice among liquid handlers is the Freedom EVO automated liquid handler from Tecan in Durham, North Carolina. Others include the Sciclone ALH Workstation from Caliper Sciences (Hopkinton, Massachusetts), the BioCube System from Proteodyne (Windsor, Connecticut) and the 925 PC Workstation and GX-281 Liquid Handler from Gilson (Middleton, Wisconsin). “You can use over 300 standard racks and many more custom ones,” says Gilson's spokesperson Greg Robinson.

Going large

Just swell: Pierce Biotechnology's SwellGell discs. Credit: PIERCE

For crystallization and other functional studies, a researcher needs more protein than is possible in 96-well plate format. For such purposes, a number of parallel-purification systems that can accommodate larger volumes of cells are starting to come onto the market.

GE Healthcare's AKTAxpress is a fully automated chromatography system for purification of His- and GST-tagged proteins, yielding up to 50 mg of target protein. Affinity chromatography is the first step of all protocols, but as a second step it is possible to choose between desalting or gel filtration, and an ion-exchange step can be added too. Automatic tag removal is possible in all multi-step protocols.

Teledyne Isco (Lincoln, Nebraska) launched the BioOptix 10 last year, a ten-channel parallel-purification system for high-capacity protein purification with subsequent fraction collection. This means it can purify protein from ten different samples by ion exchange, affinity or size-exclusion chromatography. The instrument includes a high-capacity fraction collector (20 to 60 fractions per sample). Ten independently controlled pumps can be programmed with different gradient conditions for rapid identification of columns and conditions that give optimal results. “Instead of doing it sequentially, a process that can take 4–5 days, you can load different columns and run the instrument over lunch,” says John Urh, product manager for chromatography

Another player in this arena, QIAGEN's BioRobot Protein LS System, has the capacity for the parallel purification of up to 24 large-scale cultures in less than three hours, and PerkinElmer's JANUS system can be configured for large-scale purification protocols.

New reagents and instruments have allowed researchers to set up pipelines for high-throughput protein production of a scale and capacity that match the needs of their individual labs. As a result of these advances, hundreds of proteins from different organisms have been purified and their structures and functions determined. But keeping abreast of this rapidly changing field will require ongoing innovation. “Several years ago everyone was talking about genomics, now the focus is on proteomics. More and more we are seeing research moving toward specific proteins,” says PerkinElmer's Hall. “The challenge is trying to keep up with the technologies.”