Much of the information presented in this text was gained through the study of highly purified protein molecules. To deter-mine the structure and/or functional properties of a specific pro-tein it is necessary to separate that propro-tein from the other biomol-ecules in the cell (lipids, nucleic acids, saccharides) and, of course, all other proteins. In a typical cell the protein of interest is usually a minor component; thus, the isolation of that protein from such a complex mixture presents a challenge. Modern methods of gene expression and protein purification have simplified the problem by increasing the concentration of the desired protein within the cell and exploiting specific interactions between the protein and materials used in the purification process.
Recombinant Protein Expression
To begin, let’s consider the problem of protein concentration. A typical enzyme may represent only 0.01% of the soluble protein in a cell. Thus, a 10,000-fold enrichment is necessary to purify that protein to homogeneity. If recombinant DNA technology (see Tools of Biochemistry 4B) can increase the intracellular abundance of that protein to 1%, then only a 100-fold enrichment is required;
if to 10%, then a 10-fold enrichment will suffice. Historically, pro-teins were purified from natural sources, such as animal or plant tissues. The first proteins to be studied in detail were those with high abundance in particular tissues (e.g., hemoglobin in red blood cells). For proteins that are present in low concentration in their natural tissues, it is necessary to harvest large amounts of the tissue to isolate a useful amount of the desired protein. This unfortunate scenario was a fact of life for biochemists for many years until the tools of “recombinant protein expression” were developed and widely adopted in the late 1970s and early 1980s. Recombinant protein expression allows researchers to produce proteins of inter-est at relatively high concentrations within cells and also enables the production of so-called site-directed mutants, which are pro-tein variants with designed amino acid sequence alterations (see Tools of Biochemistry 4B). Frequently, the mutant proteins exhibit changes in structure, function, and/or stability relative to the
“wild-type” (i.e., naturally occurring) protein; thus, the mutants are of great scientific interest. Another important feature of recom-binant technology is the ability to express a wide variety of foreign proteins in host cells. For example, many proteins of animal or plant origin have been successfully expressed in Escherichia coli cells. Because E. coli can be easily programmed to produce foreign proteins and they grow quickly compared to most plants and ani-mals, E. coli cells can be viewed as convenient “factories” for protein production; however, E. coli cells are limited in the types of post-translational modifications they carry out. It is usually necessary to express proteins in eukaryotic systems if some post-translational modification (e.g., glycosylation) is required for activity.
Recombinant protein expression technology is based on the observation that the amino acid sequence for a protein is deter-mined by the sequence of the DNA in the gene that encodes that protein, as described in Chapters 4 and 5 (see Figures 4.23, page 112, and 5.18, page 152). In theory, any protein sequence can be expressed in a cell that contains a copy of the gene encod-ing that protein. E. coli can be made to take up small circular DNA molecules, called “expression vectors”, that are on the order of 2–10 kilobases in length. An expression vector is a modified form of a natural extrachromosomal DNA, such as a plasmid, which is capable of autonomous replication in a bacterial cell.
Recombinant DNA technology allows a researcher to cut open that plasmid at a desired site and splice in a gene encoding the protein of interest. As shown in Figure 5A.1, the gene encoding the wild-type or mutant protein to be expressed is within each vector, along with a gene encoding a so-called selection marker.
The selection marker is usually a protein that confers resistance to an antibiotic that is included in the cell growth medium; thus, only those cells that have taken up the vector, and are thereby capable of expressing the desired protein, will survive in the growth medium. With many tens to hundreds of copies of the vector in each cell, production of the desired protein is maxi-mized. Using this approach, even those proteins that occur in low intracellular concentrations in nature can be produced in suffi-cient yield to allow biochemical characterization and/or com-mercial production.
PROTEIN EXPRESSION AND PURIFICATION
161
Protein expression vector 3098 bp
octinmleearSkergene M
yog lob
ingnee
Ori
FIGURE 5A.1
Schematic representation of a generic protein expression vector. The circle represents the double-stranded DNA sequence for the entire vector. The box marked “ori” is the “origin of replication”, which determines how many copies of the vector will be made in the cell. The arrows represent locations of protein genes in the vector DNA sequence. This vector will express two proteins: the recombinant protein of interest (red arrow, in this case, myoglobin) and a so-called selection marker (green arrow).
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CHAPTER 5 INTRODUCTION TO PROTEINS: THE PRIMARY LEVEL OF PROTEIN STRUCTUREThe Purification Process
Although recombinant technology can increase the concentra-tion of a specific protein inside the cell, the problem of separating the desired protein from all the other cellular components remains. The sequence of steps taken to purify a given protein will be unique to that protein because proteins vary in chemical properties; however, many features of the purification process are common. For example, most purification steps begin with lysis, or rupture, of the cells. Cell lysis can be achieved by sonic disrup-tion (“sonicadisrup-tion”), mechanical rupture using a homogenizer, or by enzymatic digestion of the cell wall. This is followed by cen-trifugation to remove unbroken cells and insoluble cell parts (e.g., membranes) to yield an extract, called the “cell lysate,”
which contains the soluble proteins and other biomolecules in a cell. The desired protein is then purified from the other proteins in the lysate by one or more of the following commonly used steps: (1) affinity chromatography, (2) ion exchange chromatog-raphy, or (3) size exclusion chromatography. As illustrated in Figure 5A.2, purification of the desired protein by chromatogra-phy is the result of differential interactions between the various proteins in the mixture loaded onto the chromatography column and the matrix within the column. In general, the more strongly some protein interacts with the matrix, the later it will elute from the column. Proteins are generally detected by UV absorbance at 280 nm, or 220 nm, as they elute from the column.
Affinity Chromatography
Affinity chromatography relies upon selective adsorption of a pro-tein to a natural or synthetic ligand, typically a substrate or inhibitor, which is immobilized by covalent attachment to an inert solid support. The support that displays the bound ligand is called the “affinity matrix.” The interactions between the desired protein
and the affinity matrix are expected to be highly specific; thus, most of the contaminants in the mixture will not interact with the affinity matrix, whereas the desired protein is expected to bind tightly. As shown in Figure 5A.3, when a complex mixture flows through the affinity matrix the desired protein will bind tightly and remain bound until most contaminants are washed through the column. The bound protein can then be eluted using a variety of methods that preserve the structure and activity of the protein.
In some exceptional cases the elution methods require extreme conditions to disrupt the bonds between the protein and the matrix, including denaturation of the protein.
Specific, complementary, noncovalent interactions are the basis for much interesting biological chemistry, including antibody binding, enzyme-substrate recognition, enzymatic catalysis, gene regulation, cell signaling, and muscle contraction, just to name a
1 2 3 4 5 1 2 3 4 5 1 2 3 4 5
Buffer Buffer
Adsorbent material
Mixture of molecules Later time
Buffer Buffer
Later time
FIGURE 5A.2
The principle of column chromatography.
Affinity tag displayed on affinity matrix
Affinity matrix
Contaminating protein does not bind affinity tag Contaminating protein does not bind affinity tag Desired protein binds affinity tag
Complex mixture of proteins is loaded onto the affinity column
(a)
Contaminating proteins flow through;
the desired protein binds tightly and remains on the affinity column
Protein absorbance
Begin sample application (b)
Change to elution buffer The vast majority of proteins
don’t bind affinity matrix; thus, they are washed off the column
After nonbinding proteins are washed away, the desired protein can be eluted from the affinity matrix with “elution buffer”
Volume of mobile phase added to the column FIGURE 5A.3
(a) A simplified view of affinity chromatography. Specific binding of the desired protein (blue shapes) to the affinity matrix is shown. The contaminating proteins (green shapes, beige circles) wash through the column without binding, resulting in a significant purification of the desired protein. The bound protein can then be eluted by any one of several methods discussed in the text. (b) A schematic representation of an affinity chromatogram. The proteins that don’t bind the affinity matrix are washed off and elute early, giving rise to a large protein absorbance. After the contaminants are washed off, the desired protein is eluted, giving a smaller protein absorbance.
PROTEIN EXPRESSION AND PURIFICATION
163
ogy not only for increasing protein concentration in cells, but also for improving the protein-purification process. One possible limitation to this method is that the resulting protein sequence carries some modification compared to the wild-type, in this case, an extra six histidine amino acids.
Affinity chromatography is so efficient that in many cases no further purification steps are required; however, further steps may be required to achieve separation of the desired protein from those contaminants that are closely related (e.g., proteins that dif-fer by post-translational modifications).
Ion Exchange Chromatography (IEC)
Ion-exchange chromatography is used to separate molecules on the basis of their electrical charge. The strength of interaction between a protein molecule and an ion exchange matrix depends on (1) the charge density on the protein and (2) the ionic strength of the mobile phase, which is always a buffered solution.
The charge density on the protein is modulated by altering the pH of the solution (see Chapter 2). Recall that the overall charge on a protein is zero when the pH of a protein solution is equal to the isoelectric point (pI) for the protein, and that as the pH of a protein solution increases, the charge on the protein molecules becomes increasingly negative. Conversely, as the pH of the solu-tion decreases, the charge on the protein molecules becomes increasingly positive. This behavior is a consequence of the fact that the ionizable groups on the surface of a protein are either carboxylic acids or amines (see Problem 15 in Chapter 2).
There are two main types of ion exchange matrices: (1) anion exchangers such as diethylaminoethyl (DEAE) cellulose and quater-nary ammonium (“Q”) resins, which carry a positive charge and therefore bind to negatively charged proteins, and (2) cation exchangers such as carboxymethyl (CM) cellulose and sulfonic acid (“S”) resins, which carry a negative charge and bind to positively charged proteins. DEAE and CM exchangers are considered “weak”
ion exchangers because they carry functional groups that can lose their charges at (DEAE) or (CM). The “Q”
and “S” resins are effectively always charged in aqueous buffers;
thus, they are considered “strong” ion exchangers. It is critical to match the IEC resin and buffer to the pI of the protein of interest.
For example, a protein will bind to a column of DEAE-cellulose when the pH of the mobile phase is above the pI for that protein, but will not bind the column if buffer pH 6 pI.
pH 6 ~4 pH 7 ~10
few examples. Early in the development of affinity purification methods, many investigators immobilized the natural binding tar-gets (“ligands”) for a protein on a solid chromatography support to take advantage of such specific binding interactions. A related tech-nique, called “immunoaffinity” chromatography, takes advantage of the high binding specificity of antibodies for their ligands.
Antibodies raised against a given protein can be covalently attached to a solid support to make an affinity matrix that binds selectively and reversibly to that protein. Although immunoaffinity columns are efficient, the costs and time required to produce the antibodies are significant; thus, this method has largely given way to faster and less expensive techniques.
One of the most prevalent affinity methods, immobilized metal affinity chromatography (IMAC), takes advantage of the strong interactions between a or ion and a string of six sequential histidine residues. The amino acid sequence (His)6is called a “hexahistidine-tag,” (or “His-tag”) and it is quite rare in nature; thus, few naturally occuring proteins will bind to the IMAC matrix. Using recombinant DNA technology, the His-tag can be appended to the gene encoding the desired pro-tein, as shown in Figure 5A.4. When this protein is expressed it will include the His-tag sequence and therefore it will bind tightly to the IMAC matrix. The bound protein can be eluted from the IMAC column with a buffer containing imidazole (an analog of the His side chain), or a low pH buffer (which protonates the His side chains and reduces metal ion binding), or with a buffer con-taining the metal chelator EDTA. EDTA effectively removes the metal ion from the column and thereby disrupts the bonding interactions between the protein and the affinity matrix.
The His-tag affinity method described above is used exten-sively to produce purified proteins for biochemical characteriza-tion. This example illustrates the power of recombinant
technol-Co2+ Zn2+,
Ni2+,
Vector Vector
DNA encoding protein DNA encoding protein
Ligation
HIS-T AG HIS-T
AG
FIGURE 5A.4
Insertion of a protein gene into one of several commercially available expression vectors results in addition of the affinity “His-tag” (i.e., a sequence of six His residues) to the protein sequence.
Weak anion exchanger (DEAE) Strong anion exchanger ("Q")
Weak cation exchanger (CM) Strong cation exchanger ("S") N
H (X)n
N R R (X)n R
(X)n O
S O O (X)n O−
O−
+ +
Elution of a protein bound to an IEC matrix can be achieved by changing the pH of the mobile phase such that the charge on the
164
CHAPTER 5 INTRODUCTION TO PROTEINS: THE PRIMARY LEVEL OF PROTEIN STRUCTUREprotein is reduced, thereby weakening its binding to the support and/or increasing the ionic strength of the mobile phase (e.g., by addition of some salt to the elution buffer). Soluble ions compete for binding to the charged functional groups on the matrix. As the concentration of soluble ions increases, the ions will out-compete, and thereby displace, the protein from the matrix.
In summary, IEC allows for separation of proteins based on differences in charge density. The theoretical basis for this separa-tion technique was presented in Chapter 2 and includes the fol-lowing concepts: (1) relative strengths of electrostatic interactions, as described by Coulomb’s law, (2) the isoelectric point or pI for a given protein, and (3) modulation of charge density on a protein as a function of pH, as described by the Henderson–Hasselbalch equation.
Size Exclusion Chromatography (SEC)
Size exclusion chromatography, which is also known as “gel filtra-tion chromatography,” differs from the two methods above in that noncovalent interactions between the protein and support are neg-ligible. As shown in Figure 5A.5, SEC separates proteins on the basis of apparent size, or “hydrodynamic radius.” The apparent size of a protein molecule is approximately correlated with the length of the protein amino acid sequence. This rule of thumb assumes, to a first approximation, that soluble folded proteins behave as spheres. The distinguishing feature of the SEC stationary phase is the porous structure of the matrix (Figure 5A.5b). These matrices are generally spherical beads with pores in the surface of the bead that are on a size scale close to that of protein molecules. Different SEC matrices have larger or smaller sized pores in the beads.
The principles at work in SEC are diffusion by Brownian motion and “excluded volume.” The total volume in an SEC col-umn includes the volume of the porous beads and the volume of the mobile phase, which is in the space between the beads and also inside the beads. The volume of buffer solution required to elute a protein from the column depends on what fraction of the column
volume that protein can occupy: the more volume the protein can occupy, the more buffer will be needed to elute the protein, and the later it will elute from the column. The size of the pores in the beads determines which proteins can occupy volume inside the bead and which proteins cannot. Because smaller proteins are more likely to diffuse into the interior of a bead, they will occupy more of the total column volume than a large protein, which can only occupy the volume between—but not inside—the beads. In other words, larger proteins are “excluded” from the volume found inside the beads, and, consequently, larger proteins elute from an SEC column earlier than smaller proteins. Note that this order of elution by size is the reverse of the order of migration in an SDS-PAGE experiment (see Tools of Biochemistry 2A).
SEC works best as a purification technique when the differ-ences in size between the desired protein and the contaminants are a factor of two or greater; thus, for SEC to be effective the complexity of the protein mixture must be relatively low. For this reason SEC is often the last step of a purification process. In addi-tion, SEC is a convenient way to “desalt” or change the buffer com-position of a protein solution because the salts that make up a buffer are low molecular weight and elute well after the desired protein. For example, it is convenient to run affinity columns and IEC columns in nonvolatile buffers such as phosphate-buffered saline (PBS); however, nonvolatile buffers are not compatible with lyophilization (freeze-drying) and many mass spectrometry tech-niques. Mass spectrometry is a powerful tool for protein analysis and is often used to confirm the identity of a purified protein (see Tools of Biochemistry 5B). To prepare a protein for lyophilization or mass spectrometry, an SEC column is equilibrated with a mobile phase that uses a volatile buffer, such as ammonium acetate. As a protein, which is loaded onto the SEC column in PBS, moves through the column it becomes separated from the phos-phate buffer salts and is surrounded instead by the ammonium acetate buffer solution. In this way a “buffer exchange” from PBS to ammonium acetate occurs, and the protein is now ready for lyophilization and/or mass spectrometric analysis.
Small molecules can penetrate beads; passage is retarded
Column of stationary porous beads
Large molecules move between beads Solvent flow
(a) (b)
FIGURE 5A.5
The principle of size-exclusion chromatography.(a) As a mixture of proteins flows down the column, the smaller proteins are retarded; thus, the larger proteins elute first. (b) A close-up of an SEC bead (gray). Proteins larger (green) than the pores in the beads flow around the beads; but, the smaller pro-teins (red) can enter the pores in the beads, resulting in retarded mobility through the column.
PROTEIN EXPRESSION AND PURIFICATION
165
Example: Purification of a Recombinant Myoglobin Mutant
Figure 5A.6 illustrates a scheme for the purification of a mutant form of the eukaryotic muscle protein myoglobin expressed in E. coli bacteria. This particular mutant includes a 16 amino acid insertion into the protein sequence. Following the period of protein production in bacterial cells, the cells are lysed so that the contents of the cytoplasm are released into a buffered solution. The soluble material can then be separated from the insoluble material (e.g., membranes, precipitated protein aggregates) by centrifuga-tion. The resulting supernatant is a complex mixture of nucleic acids and proteins, as shown in Lane 1 of Figure 5A.7. An efficient purification scheme will significantly reduce the complexity of the mixture in the early steps of the process; thus, affinity chromatog-raphy is a good choice for the first chromatogchromatog-raphy step. In this case an IMAC purification step achieves significant purification of the mutant myoglobin, as shown in Lane 2 of Figure 5A.7. After IMAC, all the remaining contaminants appear to be of higher molecular weight than the mutant myoglobin; thus, SEC can be
used to separate the mutant from the contaminants, as shown in Lanes 3 and 4 of Figure 5A.7. This is an efficient purification scheme because only two chromatography steps are required to achieve greater than 95% homogeneity for the desired protein.
References
Janson, J.-C., and L. Rydén (1998) Protein Purification: Principles, High Resolution Methods and Applications (2nd ed.). Wiley-VCH, New York.
Roe, S. (ed.) (2001) Protein Purification Techniques: A Practical Approach (2nd ed.). Oxford University Press, Oxford.
Rosenberg, I. M. (2005) Protein Analysis and Purification: Benchtop Techniques (2nd ed.). Birkhauser, Boston.
Scopes, R. K. (1994) Protein Purification: Principles and Practice (3rd ed.).
Springer, New York.
FIGURE 5A.7
The myoglobin mutant is purified to greater than 95% homogeneity by a two-column procedure. A Coomassie-stained 15% SDS-PAGE gel is shown. Lane 1:
E. coli lysate (after centrifugation). Lane 2: IMAC purified proteins; the prominent band is the desired myoglobin mutant. Lane 3: 2 g of the mutant myoglobin after SEC. Lane 4: 10 g of the mutant myoglobin after SEC. Lane 5: SEC-purified wild-type myoglobin. Lane 6: Protein molecular weight markers.
Reprinted with permission from Biochemistry 41:13318–13327, A. L. Fishburn, J. R. Keeffe, A. V. Lissounov, D. H. Peyton, and S. J. Anthony-Cahill, A circularly permuted myoglobin possesses a folded structure and ligand binding similar to those of the wild-type protein but with a reduced thermodynamic stability.
© 2002 American Chemical Society.
m m
Recombinant Myoglobin Purification Scheme Express protein in E.coli cells
Lyse cells, separate solids from lysate by centrifugation
Soluble fraction Insoluble fraction (discarded)
Affinity chromatography
(IMAC)
Size exclusion chromatography
(SEC) FIGURE 5A.6
Flow chart for the purification of recombinant myoglobin.
1 2 3 4 5 6 kDa
14 22 31 45 66