Liquid Chromatography: Products in the Protein Chemist's Tool Chest
Liquid chrom-atography is to the protein re-searcher what the hammer is to the carpenter. Liquid chromatographic techniques comprise a major portion of the repertoire of protocols the protein researcher can call upon to purify and analyze proteins. Modern liquid chromatography has come a long way since its infancy-when early matrices were capable of providing only crude separations-to modern matrices and technologies that can accomplish the purification of a protein to homogeneity in a single
Mar 16, 1998
Christopher Smith
Liquid chrom-atography is to the protein re-searcher what the hammer is to the carpenter. Liquid chromatographic techniques comprise a major portion of the repertoire of protocols the protein researcher can call upon to purify and analyze proteins. Modern liquid chromatography has come a long way since its infancy-when early matrices were capable of providing only crude separations-to modern matrices and technologies that can accomplish the purification of a protein to homogeneity in a single chromatographic step. A list of representative purification applications for matrices available today is presented in the Application Guide (see page 19). The term chromatography originates from Mikhail Tswett's work on plant pigments (chromophores) in the early 1900s. But modern liquid chromatography had its beginnings in the late 1950s with the introduction of cellulose-based ion exchangers (E.A. Petersen, H.A. Sorbet, J. Am. Chem. Soc., 78:751-755, 1956) and cross-linked dextran size exclusion media (J. Porath, P. Flodin, Nature, 83:1657-1659, 1959). With these matrices, researchers could for the first time make basic isolations of proteins from crude extracts of biological material. Since then many different matrices have been developed based on the full spectrum of physical and chemical properties of the support matrices, bound ligands, solvent systems, and protein being purified. The cornucopia of matrices utilizes surface hydrophobicity, net charge, size, antigenicity, substrate or other ligand affinities, and surface (exposed) sulfhydryl residues of the target protein(s). The introduction of new products has been relatively consistent throughout the years, but the advent of molecular biology has had a tremendous impact on hastening advances in chromatographic technologies, especially affinity-based systems. This article represents a synopsis of the chromatographic matrices available for the purification of proteins. For a more comprehensive review, see "The Guide to Protein Purification" (M.P. Deutscher, [ed], Methods in Enzymology, vol 182, Academic Press, 1990), Current Protocols in Protein Science (J.E. Coligan et al., John Wiley & Sons, 1995), or Protein Purification Protocols (S. Doonan, [ed.], Humana Press, 1996). Although some mention will be made of prepacked media, the major focus will be on bulk media researchers can use to pack their own columns. In addition, columns and media for analytical research (molecular size determination, quantitation, and/or small molecule identification), bioprocessing, and high pressure liquid chromatography will not be emphasized.
Most, if not all, protein purification schemes involve at least one liquid chromatographic step. The number of steps, matrices utilized, and order depend largely upon the physicochemical properties of the protein of interest and the biological source of the protein. The first chromatographic step in most purification schemes is size exclusion chromatography (SEC); because of this and the simplicity of the technique, it is perhaps the most widely utilized chromatographic protocol. Protein fractionation (separation) is achieved by passage through a matrix composed of inert beads with defined porosity. Proteins in the fractionation range of the matrix are momentarily caught in the pores of the beads, slowing their movement, while smaller and larger molecules easily pass either through the pores or around the beads entirely. Some of the earliest chromatographic matrices were of this genre.
Some variation of ion exchange or affinity chromatography usually follows gel filtration, although this is not always the case. Ion exchange chromatography (IEC) is based upon the net charge of a protein at a given pH and the charge-dependent affinity between the protein and charges on a support matrix. For example, negatively charged (anionic) resins will generally bind proteins with a net positive charge. Negatively charged or neutral proteins will pass through the matrix, and positively charged proteins (with varying degrees of charge) can be discriminately eluted by gradually changing (in a linear fashion) the counterion charge of the system with a salt. Some of the first and most commonly used matrices of this class include diethylaminoethyl (DEAE) cellulose and carboxymethyl (CM) cellulose, available from most chromatographic suppliers. Recent additions to this tool chest include quaternary ammonium (Q), quaternary aminoethyl (QAE), and methyl sulphonate (S) ion exchange groups on a variety of inert matrices, such as MonoBeads (hydrophilic polyether resin) from Pharmacia Biotech or Bio-Gel A from Bio-Rad Laboratories. See Table 1 for a more complete inventory of ion exchange matrices.
 Hydroxyapatite beads: Photo courtesy of Bio-Rad Laboratories |
Hydrophobic Interaction Chromatography. Hydrophobic interaction chromatography (HIC) utilizes the attraction of a given molecule for a polar or nonpolar environment. In terms of proteins, this propensity is governed by the hydrophobicity or hydrophilicity (depending on your perspective) of residues on the exposed, outer surface of a protein. Thus, a researcher may fractionate proteins based upon their varying degrees of attraction to a hydrophobic matrix, typically an inert support with alkyl linker arms of 2-18 carbons in chain length. Various kinds of matrices of this class are offered by a number of manufacturers: Phenyl Sepharose CL-4B and Octyl Sepharose 4 from Pharmacia Biotech; Macro-Prep Methy and t-Butyl Support from Bio-Rad; and Toyopearl butyl and phenyl from TosoHaas. (Table 2)
Hydroxyapatite Chromatography. Hydroxyapatite (HAP) usually refers to the crystalline form of calcium phosphate. Protein fractionation is achieved by the nonspecific interaction of positively charged proteins with the negative charges of immobilized phosphate ions. HAP is versatile in its selectivity and resolution but has achieved only limited use in the laboratory because of its inconsistent chromatographic behavior and low binding capacity. These traits seem to have been overcome with the recent introduction of ceramic, microporous HAP developed by Bio-Rad Laboratories. In addition to Bio-Rad, CalBiochem and Sigma Biochemicals also market HAP bulk media products. (Table 3)
Chromatofocusing. Although not as popular as other chromatographic methods, chromatofocusing is another versatile, high-resolution technology for fractionation of proteins based upon their isoelectric point (pI). In a nutshell, an ion-exchange matrix is equilibrated with amphoteric buffers, a protein mixture is added to the matrix, and pI fractionation is achieved by the subsequent addition of a single polyionic buffer. As this buffer passes through the matrix it creates a descending linear pH gradient. Proteins elute from the matrix at that point in the gradient where the pH matches their pI. The only chromatofocusing system available today is that provided by Pharmacia Biotech, which includes the Mono-P column and polybuffer exchange (PBE) ampholyte buffers.
Covalent Chromatography. Another technique in the protein biochemists chest of purification tools is reversible covalent chromatography. This technique is based upon the creation of a disulfide bridge between the cysteine sulfhydryl groups of the protein and those immobilized on a support matrix. The proteins of interest are then eluted by the addition of reducing agents. Currently available covalent chromatography matrices include Activated Thiopropyl Sepharose 6B and Activated ThioSepharose 4B from Pharmacia Biotech; cysteamine agarose, cysteine agarose, and glutathionine agarose from Sigma; and N-acetylhomocysteine agarose from Bio-Rad Laboratories.
Affinity chromatography has progressed immensely from its inception, when insoluble materials such as starch, cellulose, and phosphocellulose were the only matrices available. Today, affinity media include practically any ligand immobilized on an absorbent support matrix. The natural binding affinity between a ligand (immobilized on an inert support) and a specific protein is the basis for this chromatographic technique. The ligand can be a low molecular weight compound (inhibitor, substrate, dye), a peptide, a truncated polypeptide, or another protein. This technique is a very powerful tool in that it provides a mechanism to selectively isolate a single protein from a soup of biological material. It is a very popular technique and the range of matrices can be grouped into one of many classes based on the general ligand types, which include reactive-dye, metal chelate, lectin, substrate/inhibitor ligand, immuno and Protein-A affinity chromatography. A representative sampling of the various matrices and their specific purification applications is given in Table 4. Although the table emphasizes matrices with pre-immobilized ligands, many manufacturers (for example Bio-Rad Laboratories, Pharmacia Biotech, and Sigma Biochemicals) also provide activated matrices ready for the custom attachment of a researcher's ligand of choice.
Modern developments in affinity chromatography have been hastened by the marriage of advances in chromatographic matrices and recombinant protein expression technologies. In this latest incarnation of affinity chromatographic technology, recombinant clones are engineered with additional sequence that encodes a protein with an N- or a C-terminal protein tag (precursor peptide). This tag is recognized by and specifically binds to a complementary ligand that is immobilized on a support matrix. Passing cell lysate over a column of this affinity matrix will bind, in most cases, only the protein with the tag. The result can be the single-pass homogeneous purification of the target protein. Uhlen et al. (Gene, 23:369-378, 1983) were some of the first to use this technique in the Protein A-coupled affinity purification of an recombinant protein product containing an engineered IgG Fc domain peptide. Since then numerous variations to this theme have come into practice, including maltose-binding protein (MBP) and immobilized-amylose, and streptavidin and immobized biotin. Today, perhaps one of the most extensively used variations of this technique is histidine tag peptide-metal chelate affinity binding. Immobilized metal ion affinity chromatography (IMAC) technology, originally introduced by Porath et al. (Nature, 258, 598-599, 1975) is based upon the chelate resin's capacity to specifically bind to highly charged molecules, including proteins (such as phosphoproteins). Biotechnologists have been able to capitalize on this specificity by engineering expression constructs that encode a highly charged prepeptide (hexahistidine) tag in addition to the target protein. Purification of the engineered target protein is then achieved, in most cases, by a single pass through a nickel metal chelate affinity matrix. This technology has the added advantage over other affinity technologies in that the tag interacts with a specific ligand that is not normally a part of the host cell repertoire of proteins. A sampling of IMAC suppliers is provided in Tables 4 and 5.
To simplify the process of matrix selection, while reducing potential assessment costs, many manufacturers offer sample packs of their products, for example, Bio-Rad's Protein Purification Sampler Pack (which includes a selection of ion exchange, HIC, and HAP media in 5 ml cartridges) or Pharmacia Biotech's HiTrap Ion Exchange or HIC Test Kits. For researchers who only need to purify small amounts of protein, prepacked mini-size columns are available, such as Bio-Rad's Econo-Pak cartridges or Pharmacia Biotech's Hi-Trap 1 and 5 ml columns. Buffer exchange of minute quantities of protein may also be achieved with media prepacked into microcentrifuge tubes. For those researchers who have the time and expertise, most chromatographic media are available in bulk (unpacked) form), so researchers can pack columns with matrices to their own specifications. Alternatives to bulk media are prepacked columns. These are attractive options because they reduce the researcher's investment of time, material, and effort in creating only one tool, diverting the scientist from actual research. In addition, advances in column packing technology have resulted in the commercial manufacture of columns with reproducible column-to-column fractionation characteristics (plate number, resolution, etc). The fractionation characteristics of prepacked columns are hard to match by homegrown packing, and it is easier for collaborators to contribute to the research or test prior findings using columns from a general source.
Christopher Smith is an Associate Staff Scientist at San Diego Super Computer Center and can be reached at csmith@sdsc.com.
 Tables 1-4 Table 5 |
