Affinity chromatography for protein isolation William H. Scouten Baylor University, Texas, USA Thousands of reports concerning protein purification have appeared in the past year, and over 150 of these involved, at least in part, the affinity chromatography process. Immobilized membrane affinity chromatography, temperature-programmed elution, and centrifugal affinity chromatography are among the most significant new techniques amid the myriads of applications in this mature field. Current Opinion in Biotechnology 1991, 2:37-43
Introduction Given the vast amount of literature on modes of protein purification, it would be impossible for this review to cover more than a few of the most novel procedures. Therefore, the focus of the review is directed towards affinity chromatography techniques in the purification of proteins [1.°], although major advances in protein quantification will also be considered. A review of some of the literature concerning affinity chromatographic purification of protein reveals that in approximately half the cases (49 out of 91), purifications are made using commercially prepared alfinity chromatographic media. Some of the most common applications of commercial matrices are dye chromatography (19 out of 91 cases), hydrophobic chromatography, mainly using phenyl Sepharose (12 out of 91 cases), heparin chromatography (10 out of 91 cases), lectin a/rarity chromatography (8 out of 91 cases), and various ATP, coenzyme A, and NAD derivatives (12 out of 91 cases). In addition, protein A and/or G has been used to purify immunoglobulin (Ig)G which, in tum, is frequently employed to prepare an immunoabsorbent chromatography matrix. Approximately 20 96 of all affinitychromatography applications use one or more immunoalfmity steps. When affinity chromatography was first introduced to the public, there was great enthusiasm for the possibility of a 'one-step total purification' [2]. The technique has not, however, lived up to such expectations except in those few instances where a single affinitychromatography step will suffice. One of the more striking purifications affected over the past year has been the purification of the soluble blood group A-glycosyltransfemse [3°] which can be obtained with a 770 000-fold purification in two steps, the first of which is an atfmity chromatographic step which yields a 116 000-fold purification. Most affinitychromatography purifications involve multiple steps and, frequently, several steps are employed in the same procedure. It is
not uncommon for two or even three affinity steps to be employed: a combination of dye affinityand hydrophobic affmity are the most commonly used, and these are often followed by a third separation using either heparin, lectin, or protein A chromatography. Even more frequently, a step relying upon a commercially prepared affinity matrix is coupled with a second atfinity step based upon an immobilized ligand prepared in the investigator's laboratory. The purpose of this brief overview has been to acquaint the reader with the wide variety of materials that are available commercially for application to affinity systems, and to emphasise the ease with which investigators can prepare tailor-made affinity purification materials in the laboratory. I will now describe a few novel aspects of the application of affinity chromatography to protein purification and a selected group of illustrative examples of the use of affinity materials in purification.
Affinity chromatography elution methods The key to successful affinity chromatography lies in the elution method. A wide variety of methods have been employed in the past, ranging from elution by changes in the pH [4] or salt concentration [5], to the application of an electric field through an atfinity matrix [6]. Some purifications have been extremely successful through the use of sulficient prewash to remove any protein which has nonspecifically bound to the column or which may have had an affinity for the column because of adventitious ion exchange or hydrophobic chromatography effect. This procedure is followed by the addition of an elution buffer containing free substrate or inhibitor in appropriate concentration. Frequently, the substrate must be allowed to dwell on the column for some time, as the rate of elution by free ligands can be rather slow. Bergold and Cart [7 °°] have recently introduced a
Abbreviations HPLAC--high-pressure liquid affinity chromatography; HPLC--high-pressure liquid chromatography; HSV-l--herpes simplex virus type 1; Ig--immunoglobulin. © Current Biology Ltd ISSN 0958-1669
37
38 Analytical biotechnology rather intriguing method for the elution of glycoproteins from an immobilized lectin which involves an increasing column temperature profile. This is achieved by placing a small high-pressure liquid affa~ty chromatography (HPLAC) column containing immobiliTed concanavalin A within a commercial high-pressure liquid chromatography (HPLC) mobil phase preheater. By accurately programming the column temperature, the adsorbed glycoproteins can be eluted gently and cleanly without any alteration to the chemical composition of the elution buffer. With the correct change in temperature, the dissociation of glycoprotein from the immobilized lectin occurs very rapidly and, consequently, the protein is eluted in a highly concentrated peak. It should be possible to separate many different adsorbed glycoproteins by proper control of the temperature program. Bergold and Carr [7oo] have also shown that the addition of the competitive binding agents normally used In elution of glycoprotein can have a positive effect on the rate of elution within a given temperature program. This illustrates one of the many useful variations of this technique. Because of the success of the method and the ease by which the instrumentation can be assembled, it is likely that this technique will be applied to a wide variety of other atFmity chromatographic procedures in the future. A similar methodological advance in a/finity chromatography has been achieved by Berg and myself [8.*] using centrifugal force to create a multicolumn, simultaneous centrifugal affinity chromatography procedure. In this way, a large number of immobilized dyes have been screened for their ability to bind to goat IgG and, specifically, to the Fc fragment of the IgG molecule. The procedure enables the screening of 65 different immobiliTed dyes within a few hours, suggesting that the speed with which dye materials can be screened is limited by the number of carrier places present in the centrifuge employed in the process. It is conceivable that as many as 100 different dyes could be screened simultaneously in a matter of two to three hours. A similar technique for screening dye matrices, using a dye mate and a microtiter plate, has been reported by Hondmann and Visser [9"]. Another procedure for the elution of proteins from affinity matrices which may receive wide-spread application in the future, is the discovery of a calcium-dependent antibody that binds to a specific short hydrophilic polypeptide chain in the presence of calcium, but which releases the polypeptide when calcium-chelating agents are present [10*.,11]. This technique is suitable for the 'preplanned purification' of recombinant proteins. In such cases, the gene of the protein is modified to contain a leader sequence which codes for a peptide fragment that can be employed in the purification of the resulting recombinant fusion protein on a specific chromatographic matrix (see [12] for an excellent review). For example, recombinant fusion proteins containing a polyhistidine leader sequence can be purified on a metal chelate (nickel) chromatographic matrix [13]. In the calciumdependent modification of this type of purification, as developed by Hopp [10-*,11], the calcium-binding peptide portion of the fusion protein binds to the anti-
body molecule in question in the presence of calcium in an immunoabsorbent step. Subsequent addition of a calcium chelate agent allows rapid removal of the engineered protein. The leader peptide, which is termed the 'FlagTM' segment, can subsequently be detached from the purified protein by treatment with an enterokinase that cleaves immediately after the Asp-Asp-Asp-Asp-Asp-Lys sequence of the FlagTM. This procedure is currently being marketed by Immunex Corp. Reardon [ 14°] has proposed an interesting reversal of an earlier application of affinity chromatography in which an af~nity system was used to determine whether the binding of substrates to the active site of an enzyme occurred in a mandatory sequence. Herpes simplex vires type 1 (HSV-1) DNA polymerase was bound to a matrix containing a DNA template primer with an acyclovir monophosphate residue attached to the 3' terminus. In the absence of additional nucleotides, this column behaved as a simple DNA afl'lnity matrix and DNA polymerase could be chromatographed as any other DNArecognizing protein using salt gradient elution. However, when dGTP was added to the system, the binding of the HSV-1 DNA polymerase to the affinity matrix was drastically increased and the enzyme was no longer readily eluted, even by 1 M sodium chloride. Elution could only be achieved by the removal of the dGTP from the elution buffer. This 'mechanism-based' affinity chromatography is very selective and, therefore, should be applicable to a high purification, 'one-step' affinity system for those enzymes that possess mandatory sequential binding for two or more substrates.
Matrix preparation Numerous reviews exist on the matrix selection procedure and its importance in affinity chromatography [15,16]. However, the significance of the chemical method of coupling ligands to matrices has only begun to be fully appreciated recently. Investigations into the position and orientation of ligands, especially protein ligands, have been performed using cleavable spacer arms conraining reporter functions, such as radioisotopic labels [17]. Similar reagents have been employed previously as immobilized protein modification reagents (Fig. 1) [ 18]. This procedure will certainly gain recognition as the importance of the orientation of the immobilization of protein ligands becomes more obvious. Several examples of the necessity for a proper ligand immobilization have been reported recently. One of the most interesting is the development of an oriented immobilization of protein A as an IgG alfinity matrix [19o]. The use of immobilized protein A as the coupling agent and spacer for immobilizing the antibody yields an immunoabsorbant with twice the capacity of that achieved using cyanogen bromideactivated agarose. The presumption is that protein A orients the IgG molecules by binding to their Fc portions. This forces the antigen-binding portion of the molecule into the solution, thereby increasing its binding capacity. Similarly, hydrazide agarose and cellulose can be used to
Affinity chromatography for protein isolation Scouten 39
I Cleavablearm I II
Matrix~ J ~
U
~
Attachment point
I Po'nt
Reporter~ P r o t e i n
-9,oetector I
I moiety ]
I
I-'.Pr°,te'"
lattpCnntentl
n Matrix]
I
+N
roteio I
orient periodate, or enzyme-oxidized IgG [20 o.,21.,22.. ], to increase the antigen-binding capacity of the matrix (Fig. 2).
Fig. 1. A schematicrepresentationof an immobilized protein modification reagent. Reprinted with permission from [18].
in another demonstration of the significance of the mode of ligand immobilization, Gabius [23] showed that the type of both linkage and spacer arm used to immobilize carbohydrate ligands made a significant difference to their effectiveness as an aWmitychromatography material for the purification of ~)-galactoside-binding proteins. In a similar study, three ubiquitin carboxyt-terminal hydrolases were affinity-purified by ubiquitin immobilized to a matrix via its arginine residues. In contrast, these same carboxyl terminal hydrolases were unable to recognize ubiquitin immobilized via its b/sine residues [24].
Chromatographic matrices
~Fab//'~~Fab
////v
Fab
Fig.2. Schematicdepictingthe inferreddifferencesin the orientationof antibodies immobilizedvia orientedoligosaccharidespecificchemistriesand randomamino-acid-directedchemistnes.Reprintedwith permissionfrom [21o].
One of the most interesting and novel of the chromatographic systems reported in the past year is the development of immobilized artificial membrane chromatographic materials [25"]. These matrices consist of a silica support to which a long-chain fatty add has been coupled. Normally this is done using a long dicarboxylic acid which is coupled to an amino silica surface at one end via an amide bond. Subsequently, the remaining free carboxyt is coupled to a phosphatidyt lipid to create a surface that mimics a cell membrane. Membrane proteins bind to, and are easily chromatographed on, the resulting artificial membrane surface. As membrane proteins have been notoriously difficult to handle, this material should prove to be very useful in their isolation and study. Investigation of thiophih'c absorbants as inexpensive replacements for protein A chromatography materials for IgG purification has continued; although they have not yet become wholesale replacements for protein A, it seems likely that they will do so in the near future. These resins
40 Analytical biotechnology are prepared by reacting an amino matrix with an appropriate reactive reagent containing a thiol group. For example, amino silica can be reacted with divinyl sulphone which, in turn, is treated with a large excess of mercapto-ethanol. Such a column is very efficient at purifying IgG and, for all intents and purposes, has practically the same selectivity for IgG as protein A, but without the expense and biological instability that accompanies the latter [26"]. A similar small ligand replacement medium for protein A agarose has been developed by Ngo and co-workers [27] based on pyridinyl derivatives.
Applications One of the most striking applications of affinity chromatography in the past year has been the purification of human erythrocyte acyl phosphatase [28.]. Three hundred milliliters of erythrocytes were used to purify 330 pg of the enzyme using an initial affinity chromatography step. This step, based on immunoaffinity chromatography on anti-acyl phosphatase antibodies immobilized to Sepharose 413, effected a 135 000-fold purification in a single step with a 44% yield. Subsequently, the enzyme was purified to homogeneity by gel filtration combined with reversed-phase chromatography for a total 255 000-fold purification. The immunoaffmity step yielded the greatest purification of the three steps and it can be speculated that a second irnmunoal~ty step might have created an homogeneous enzyme as easily as (or easier than) the non-affinity procedures. Another striking example of affinity chromatography was the large scale purification of vasparaginase, an enzyme with significant anti-neoplastic activity, from E r w i n i a carotovora [29"]. The enzyme was purified from 880 liters of whole fermentation broth using a three-step process. Treatment with acetone reduced the volume of the broth and subsequent chromatography on L-aspa-
ragine immobilized on Sepharose 6-Fast Flow produced an 8.5-fold purification with a recovery of 38% of the enzyme. Dialysis and further concentration of the enzyme increased its purification to 8.9-fold with a continued 38% recovery. Although the degree of purification in this case may seem insignificant compared with the 255 O00-fold obtained by the affinity step in the purification of erythrocyte acyl phosphatase, the cell-free fermentation broth that was used as the source of enzyme contained relatively small quantities of any type of contaminant. The most striking thing about this process is the scale at which the purification can be easily carried out. The L-asparagine Sepharose 6-Fast Flow column measured 25.2cm by 18cm and contained a volume of 9 liters. The cell-free extract consisted of 1400 liters of fermentation broth clarified through an inline filtration device and loaded in a reversed flow fashion in order to prevent contamination of the column during the process. Flow rate was a significant 60 liters per hour and 10-liter fractions were collected. Under such conditions, the majority of the enzyme was eluted in a single enzyme-rich fraction and 33.6 g of active protein was obtained.
Protein quantification and visualization Quantification is extremely important in any protein purification process as well as in the preparation of affinity chromatography materials that involve immobilized proteins. Gaur and Gupta [30 °-] have developed a very successful method for the estimation of amino groups on polymer supports based on the high extinction coefficient of the trityl moiety (Fig. 3). Initially, protein is trityiated using either sodium dimethoxytritytbutamte or 2-4-dinitrophenyl dimethoxytrityibutarate. The tritylation process can be controlled so that only amine functions on the polymer support are tritylated. When the excess reagents have been removed, the dimethoxytrityl cation can be released by treatment with perchloric acid. The
Fig. 3. Reactions involved in the spectrophotometric determination of solidsupported amino groups with ~'rnax= 498nm and E49a=70000M-1cm -1. DDTB, 2-4-dinitrophenyl dimethoxytritylbutarate; DMAP, dimethyl aminopyridine; DMF, dimethyl formamide; P, polymer support; SDTB, sodium dimethoxytritylbutarate. Reprinted with permission from [29".].
Affinity chromatography for protein isolation Scouten absorbance of the released cation, which has an extinction coefficient at 498 nm of 70 000 M - l c m - 1, is then measured spectrophotometrically. The high extinction coefficient of the trityl cation allows the very sensitive determination of amines on the polymer matrix. This is of obvious value in quantifying proteins immobilized to an affinity support material. Conversely, the method can be used to determine the number of amine groups on an amine-modified matrix, which is to be used for the immobilization of an affinity ligand. Another approach to protein quantification and visualization that has been exploited over the past year and is expected to become far more widely used in the future, involves recombinant fusion proteins in which protein A has been fused to an enzyme that can convert a colorless soluble substrate into a highly absorbing insoluble product. Such fusion proteins are especiaUyuseful in visualizing proteins on membranes or electrophoretic gels. The procedure involves electrophoresis of the sample followed by incubation with an antibody against the protein(s) being visualized or quantiffed. The gels are then washed and incubated with the fused enzyme-protein A. The gel is, subsequently, washed and the substrate mixture is added. After an appropriate incubation time, the protein desired (and only that protein) appears very distinctly as a colored band on the gel. This method has been demonstrated to be suitable for the quantitative deternlination of samples as small as 100 pg when protein A-neomycin phosphotransferase fusion protein is used [31o]. Similarly, the fusion protein of metapyrocatechase-protein A [32 o] has permitted the detection of bovine serum albumin in the concentration range of 0.1 n g m l - 1 to I m g m l - 1.
recent advances in biotechnology. The procedures described here, particularly temperature-programmed elution, centrifugal affinity chromatography, and the coupling of genetic engineering with affinity chromatography as 'preplanned purification' or 'affinity handles', represent major advances in the continuing development of the field. Moreover, the coming year is likely to bring as many, if not more, significant advances. Ultimately, each of these will be useful in valuable commercial and clinical applications of solid-phase biochemistry.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest oo of outstanding interest 1. oo
WU.CHEKM: 8th International Symposium on Affinity Chromatography and Biological Recognition. J OJromatogr 1990, 510:1-2. This is the lead article for the symposium issue related to the 8th International Affinity Chromatography Conference. This volume is a conpendium of the most recent advances in affinity chromatography and similar purification methods. 2.
SCOUTENWH (ed): AffiniOJ Chromatography 1981, New York: Wiley Interscience 59:1-338.
3. •
CLAUSENH, WHITE T, TAKIO K, TrrANI K, STROUD M, HOLMES E, KARKOVJ, Tram L, HAKOMORI S: Isolation o f Homogeneity and Partial Characterization of a Histo-Blood Group A Defined Fuc~tl --*2Galctl ---,3-N-Acetylgalactosaminyltransferase from Human Lung Tissue. J Bid Chem 1990, 265(2):1139-1145. This is an excellent, but not representative, example of m o d e m applications of affinity chromatography to the purification of proteins present in trace amounts in a given sample. 4.
STEERSE JR, CUATRECASASP: [3-Galactosidase. Methods gnzymol 1974, 34:350-358.
Affinity handles
5.
WEISSBACHA, POONIAN M: Nucleic Acids Attached to Solid Matrices. Methods Enzymol 1974, 34:463-475.
Downstream processing has often been the major bottleneck in the industrial production of proteins. However, during the last 5 years, a novel protein purification technique has been developed that is based on gene-fusion technology. A DNA fragment encoding an additional potypeptide fragment is fused to either the 5' or 3' end of the gene of interest (for an excellent review see [33]). The properties of the additional polypeptide tail can be taken advantage of during purification of the obtained protein product. A number of genes specifically modified to simplify protein purification have been described. Recently, such 'affinity handles' have been used to facilitate hydroprobic [34], covalent [35] and metal chelate separations [36].
6.
GRENOTC, CUILLERONCY: Isolation of Antibodies to Steroid Hormones by Affinity Chromatography on Antigen-Linked Sepharose: an Efficient Electrophoretic Elution Procedure. Bioclx,m Biophys Res Commun 1977, 79:274-279.
Conclusion Affinity chromatography and the related aspects of solidphase biochemistry have become significant elements in
7. •.
BERGOLDAF, CARR PW: Improved Resolution of Glycoproteins by Chromatography with Concanavalin A Immobilized on Microparticulate Silica via Temperature Programmed Elution. Anal Chem 1989, 61:1117-1128. This is an excellent description of an extremely novel technique for the elution of proteins from lectin columns using a temperature-programmed elution method. This should be applicable to a wide variety of affinity chromatographic systems, other than lectin/glycoprotein systems, and may well find its place in many future investigations. 8. BERG A, ScotrmN WH: Dye-Ligand Centrifugal Affinity Chro•. matography. Bioseparation 1990, 1:23-31. This describes a method for screening many different types of columns using a single sample type. In this sense, it is complementary to, and the reverse of, HPLAC. In the article, various dyes are screened for their ability to bind the Fc fragment of IgG. Such dyes could be of considerable importance in antibody purification, immobilization and application in enzyme-linked immunosorbent assays. 9. ,
HONDMANNDHA, VlSSERJ: Screening Method for Large Numbers of Dye-Absorbents for Enzyme Purification. J t2bro~ matogr 1990, 510:155-164.
41
42
Analytical biotechnology This paper presents a rapid method for screening affinity ligands using microtiter plates. Many dye-ligands can be screened for their ability to bind protein rapidly. The system is particularly applicable to enzymes with chromophoric substrates. PRICKETTKS, AMBERGDC, HOPP TP: A Calcinm-Dependent Antibody for Identification and Purification of Recombinant Proteins. BtoTechniques 1989, 7:580-589. This is an introduction to a metal ionM_ependent immunosorbant that can be used for the purification of recombinant proteins that do not yield readily to classic purification methods.
rain to hydrazide cellulose. The technique is also demonstrated to be extremely useful in the preparation of an absorbant for concanavalin A and similar lectins. 23.
GAmUSH-J: Influence of Type of Linkage and Spacer on the Interaction of [~-Galactoside-Binding Proteins with Immobilized Affinity Ligands. Aria/B/ochem 1990, 189:91--94.
24.
DUERKSEN-HUGHESPJ, WILLIAMSONMM, WILKINSON KD: Affinity Chromatography Using Protein Immobilized via Arginine Residues: Purification of Ubiquitin Carboxyi-Terminal Hydrolases. B/oc.bem/ary 1989, 28:8530--8536.
10. ..
11.
HoPP TP, PRICKETr KS, PRICEVL, LIBBYRT, MARCHCJ, CERRETrI DP, UROALDL, CONLON PJ: A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification. B ~ . b n o / o g y 1988, 6:1204-1210.
12.
SASSENFELDHM: Engineering proteins for purification. Trends Bk:~chno/1990, 8:88-93.
13.
I~GRICESFJ, GRUNINGER-LErlX=HF: Rapid Purification of Homodimer and Heterodimer HIV-1 Reverse Transcriptase by Metal Chelate AfBnity Chromatography. Eur J Biochem 1990, 187:307--314.
14. •
REARDONJE: Herpes Simplex Virus Type 1 DNA Polymerase: Mechanism-Based AtFmiW Chromatography. J B~/ OJem 1990, 265:7112-7115. This paper describes a novel method of p r o m elution by means of 'mechanism-based' affinity chromatography. The technique will be very useful in those cases where mandatory sequential substrate binding kinetics exist and where appropriate substrates and inhibitors can be obtained. It may not be widely applicable buL where it can be employed, it will be extremely effect~e. 15.
NARAYANANSP,, CRANELJ: Affinity Chromatography Supports: a Look at Performance Requirements. Trends Biotechnol 1990, 8:12-16.
16.
SCOUTENWH: A Survey of Enzyme Coupling Techniques. Methods Enzymol 1987, 135:30-65.
17.
MARBURGS, FIANAGANME, TOLMANRI, RICHARDI~ Chemistry of Solid Supports: Defining Events and Titers by Use of Cleavable, As,sayable Linking Molecules. Anal Biochem 1989, 181:242-249.
18.
LEWISC, SCOtrlEN WH: Immobilized Protein Modification Reagents. In SO//,/Phase B ~ / s t r y edited by Scouten WH. New York: Wiley interscience, 1983, pp 665-678.
19. •
AVIIADM, KAUSHALV, BARNESLD: Immunoaffmity Chromatography of Diadenosine 5'5"-PlJ~4-Tetraphosphate Phosphorylase from Saccbaromyces cerevtstae. Biotechnol Appl
B/oc/aem 1990, 12:276-283. This is an excellent example of the oriented immobilization of antibodies producing a significantly enhanced capacity when contrasted to classically prepared immunosorbams.
25. .
MARKOWCHRJ, STEVENSJM, PIDGEON C: Fourier Transform Infrared Assay of Membrane Lipids Immobilized to Silica: Leaching and Stability of Immobilized Artificial MembraneBonded Phases. Aria/Bt~bem 1989, 182:237-244. This is the latest in a series of papers describing the preparation, application, and characterization of immobilized artificial membrane afBnity matrices. Such matrices appear to be superior in the isolation of membrane-bound proteins. 26. •
NOPPERB, KOHEN F, WILCHEK M: A Thiophilic Adsorbeflt for the One-Step High-Performance Liquid Chromatography Purification of Monoclonal Antibodies Anal Biocbem 1989, 180:66-71. This papers describes an excellent recent application of thiophilic adsorbents for the purification of antibodies. This method may well replace protein A chromatography. 27.
NGO Tr, KHATFERN: Chemistry and Preparation of Affinity Liganda Useful in Immunoglobulin Isolation and Serum Protein Separation. J Oyroma~gr 1990, 510:281-291.
28. .
DEGL'INNOCENTID, BImTI A, STEFANIM, LtGUR1G, RAMPONIG: Immunoamnity Purification and Immunoassay Determination for Human Erythrocyte Acylphosphatase. B~,,c,b ~ p l BR~c/'~m 1990, 12:450-459. A report of an exceptional application of afBnity chromatography for the purification of an enzyme present in trace quantities. 29.
LEE S-M, WROBLEMH, ROSSJT: L-Asparaginase from E n v t n t a carotovorat an Improved Recovery and Purification Process Using Affinity Chromatography. ~ B/ocbem BR~tecb no/1989, 22:1-11. L-Asparaginase is purified from 880 liters of fermentation broth. This is an excellent presentation of large scale enzyme purification by atBuity chromatography for eventual biomedical and therapeutic application. •.
30. ••
GAURRig, GUPTAKC: A Spectrophotometric Method for the Estimation of Amino Groups on Polymer Supports. Anal B/ocbem 1989, 180:253-258. A very sensitive method for determination of the amine groups on immobilized matrices has been needed for a long time. This paper reports such a method.
20. •.
HUNGER H-D, SCHMIITr G, FIACHMEIER C, BEHRENDT G, COUTELLEC: High-Sensitivity Protein Detection by a New 'Contact-Copy' Method Using a Protein A-Neomycin Phosphotransferase II Fusion Protein. Ana/ B/c~bem 1990, 186:159-164. This is one of many recombinant protein A-enzyme fusion proteins being used for the visualization and quantification of antibodies and/or proteins recognized by antibodies. In general, the method aliows very sensitive quantification and visualization of specific proteins and should prove to be exceptionally useful in future investigations.
21.
32. •
22. **
33.
DOMENPL, NEVENSJR, MAIJ~ AK, HERMANSONGT, KI.ENKDC: Site-Directed Immobilization of Protein. J Cbroma~gr 1990, 510:293-302. This paper compares several commercial activated affinity chromatographic matrices with regard to their ability to immobilizeIgG and prepare affective immunosorbant matrices. O'S~NNESSYDJ: Review: Hydrazido-Derivatized Supports in Affinity Chromatography. J GM~matogr 1990, 510:13-21. ~ns' is a comprehensive review of the use of hydmzides for the immobilization of giycoproteins and other aldehyde-containing, or potentially aldehyde-containin~ molecules. TLmKOVAJ, PETKOV I~ SAJE~KJ, KAS J, BENES MJ: Carbohydrates as a Tool for Oriented Immobilization of Antigens and Antibodies. J ~ 1990, 500:585-593. This article details the preparation of a biospecific immunosorbant for ovalbumin antibodies prepared by coupling period,ate-oxidized ovalbu-
31. •
KOBATAKEE, NISHIMOmY, IKARIYAMAY, AIZAWAM, KATO S: Appfication of a Fusion Protein, Metapyrocatechase/Protein A, to an Enzyme Immunoassay. Anal BiocT~om 1990, 186:14-18. Like [31.], this paper describes an application of a protein A-enzyme fusion protein, but using a different chromophore and enzyme system for color development. SASSENFELDHM: Engineering Proteins for Purification. Trends BiotecJTno/1990, 8:88-93.
34.
PERSSON M, BERGSTRAND M, B~LOW L, MOSBACH K: Enzyme Purification by Genetically Attached Polycysteine
Affinity chromatography for protein isolation Scouten
35.
Loat~ G, PERSSONM, BOLOWI, MOSBACHI~ Metal Affinity Prccipitation of Protei~ Carryin~ Genetically Attached Polyhistidine Affinity Tails. Eur J Biochera~ 1991, in press.
and Polyphenylalanine Affinity Tails. Anal Biochem 1988, 172:3~0--337.
36.
PERSSON M, BOLOW L, MOSBACH K: Purification and Sitespecific Immobilization of Genetically Engineered Glucose Dehydrogenase on Thiopropyl-Sepharose. FEBS Lett 1990, 270:41-44.
WH Scouten, Department of Chemistry, Bayior University, Waco, Texas 76798, US&
43
Introduction Given the vast amount of literature on modes of protein purification, it would be impossible for this review to cover more than a few of the most novel procedures. Therefore, the focus of the review is directed towards affinity chromatography techniques in the purification of proteins [1.°], although major advances in protein quantification will also be considered. A review of some of the literature concerning affinity chromatographic purification of protein reveals that in approximately half the cases (49 out of 91), purifications are made using commercially prepared alfinity chromatographic media. Some of the most common applications of commercial matrices are dye chromatography (19 out of 91 cases), hydrophobic chromatography, mainly using phenyl Sepharose (12 out of 91 cases), heparin chromatography (10 out of 91 cases), lectin a/rarity chromatography (8 out of 91 cases), and various ATP, coenzyme A, and NAD derivatives (12 out of 91 cases). In addition, protein A and/or G has been used to purify immunoglobulin (Ig)G which, in tum, is frequently employed to prepare an immunoabsorbent chromatography matrix. Approximately 20 96 of all affinitychromatography applications use one or more immunoalfmity steps. When affinity chromatography was first introduced to the public, there was great enthusiasm for the possibility of a 'one-step total purification' [2]. The technique has not, however, lived up to such expectations except in those few instances where a single affinitychromatography step will suffice. One of the more striking purifications affected over the past year has been the purification of the soluble blood group A-glycosyltransfemse [3°] which can be obtained with a 770 000-fold purification in two steps, the first of which is an atfmity chromatographic step which yields a 116 000-fold purification. Most affinitychromatography purifications involve multiple steps and, frequently, several steps are employed in the same procedure. It is
not uncommon for two or even three affinity steps to be employed: a combination of dye affinityand hydrophobic affmity are the most commonly used, and these are often followed by a third separation using either heparin, lectin, or protein A chromatography. Even more frequently, a step relying upon a commercially prepared affinity matrix is coupled with a second atfinity step based upon an immobilized ligand prepared in the investigator's laboratory. The purpose of this brief overview has been to acquaint the reader with the wide variety of materials that are available commercially for application to affinity systems, and to emphasise the ease with which investigators can prepare tailor-made affinity purification materials in the laboratory. I will now describe a few novel aspects of the application of affinity chromatography to protein purification and a selected group of illustrative examples of the use of affinity materials in purification.
Affinity chromatography elution methods The key to successful affinity chromatography lies in the elution method. A wide variety of methods have been employed in the past, ranging from elution by changes in the pH [4] or salt concentration [5], to the application of an electric field through an atfinity matrix [6]. Some purifications have been extremely successful through the use of sulficient prewash to remove any protein which has nonspecifically bound to the column or which may have had an affinity for the column because of adventitious ion exchange or hydrophobic chromatography effect. This procedure is followed by the addition of an elution buffer containing free substrate or inhibitor in appropriate concentration. Frequently, the substrate must be allowed to dwell on the column for some time, as the rate of elution by free ligands can be rather slow. Bergold and Cart [7 °°] have recently introduced a
Abbreviations HPLAC--high-pressure liquid affinity chromatography; HPLC--high-pressure liquid chromatography; HSV-l--herpes simplex virus type 1; Ig--immunoglobulin. © Current Biology Ltd ISSN 0958-1669
37
38 Analytical biotechnology rather intriguing method for the elution of glycoproteins from an immobilized lectin which involves an increasing column temperature profile. This is achieved by placing a small high-pressure liquid affa~ty chromatography (HPLAC) column containing immobiliTed concanavalin A within a commercial high-pressure liquid chromatography (HPLC) mobil phase preheater. By accurately programming the column temperature, the adsorbed glycoproteins can be eluted gently and cleanly without any alteration to the chemical composition of the elution buffer. With the correct change in temperature, the dissociation of glycoprotein from the immobilized lectin occurs very rapidly and, consequently, the protein is eluted in a highly concentrated peak. It should be possible to separate many different adsorbed glycoproteins by proper control of the temperature program. Bergold and Carr [7oo] have also shown that the addition of the competitive binding agents normally used In elution of glycoprotein can have a positive effect on the rate of elution within a given temperature program. This illustrates one of the many useful variations of this technique. Because of the success of the method and the ease by which the instrumentation can be assembled, it is likely that this technique will be applied to a wide variety of other atFmity chromatographic procedures in the future. A similar methodological advance in a/finity chromatography has been achieved by Berg and myself [8.*] using centrifugal force to create a multicolumn, simultaneous centrifugal affinity chromatography procedure. In this way, a large number of immobilized dyes have been screened for their ability to bind to goat IgG and, specifically, to the Fc fragment of the IgG molecule. The procedure enables the screening of 65 different immobiliTed dyes within a few hours, suggesting that the speed with which dye materials can be screened is limited by the number of carrier places present in the centrifuge employed in the process. It is conceivable that as many as 100 different dyes could be screened simultaneously in a matter of two to three hours. A similar technique for screening dye matrices, using a dye mate and a microtiter plate, has been reported by Hondmann and Visser [9"]. Another procedure for the elution of proteins from affinity matrices which may receive wide-spread application in the future, is the discovery of a calcium-dependent antibody that binds to a specific short hydrophilic polypeptide chain in the presence of calcium, but which releases the polypeptide when calcium-chelating agents are present [10*.,11]. This technique is suitable for the 'preplanned purification' of recombinant proteins. In such cases, the gene of the protein is modified to contain a leader sequence which codes for a peptide fragment that can be employed in the purification of the resulting recombinant fusion protein on a specific chromatographic matrix (see [12] for an excellent review). For example, recombinant fusion proteins containing a polyhistidine leader sequence can be purified on a metal chelate (nickel) chromatographic matrix [13]. In the calciumdependent modification of this type of purification, as developed by Hopp [10-*,11], the calcium-binding peptide portion of the fusion protein binds to the anti-
body molecule in question in the presence of calcium in an immunoabsorbent step. Subsequent addition of a calcium chelate agent allows rapid removal of the engineered protein. The leader peptide, which is termed the 'FlagTM' segment, can subsequently be detached from the purified protein by treatment with an enterokinase that cleaves immediately after the Asp-Asp-Asp-Asp-Asp-Lys sequence of the FlagTM. This procedure is currently being marketed by Immunex Corp. Reardon [ 14°] has proposed an interesting reversal of an earlier application of affinity chromatography in which an af~nity system was used to determine whether the binding of substrates to the active site of an enzyme occurred in a mandatory sequence. Herpes simplex vires type 1 (HSV-1) DNA polymerase was bound to a matrix containing a DNA template primer with an acyclovir monophosphate residue attached to the 3' terminus. In the absence of additional nucleotides, this column behaved as a simple DNA afl'lnity matrix and DNA polymerase could be chromatographed as any other DNArecognizing protein using salt gradient elution. However, when dGTP was added to the system, the binding of the HSV-1 DNA polymerase to the affinity matrix was drastically increased and the enzyme was no longer readily eluted, even by 1 M sodium chloride. Elution could only be achieved by the removal of the dGTP from the elution buffer. This 'mechanism-based' affinity chromatography is very selective and, therefore, should be applicable to a high purification, 'one-step' affinity system for those enzymes that possess mandatory sequential binding for two or more substrates.
Matrix preparation Numerous reviews exist on the matrix selection procedure and its importance in affinity chromatography [15,16]. However, the significance of the chemical method of coupling ligands to matrices has only begun to be fully appreciated recently. Investigations into the position and orientation of ligands, especially protein ligands, have been performed using cleavable spacer arms conraining reporter functions, such as radioisotopic labels [17]. Similar reagents have been employed previously as immobilized protein modification reagents (Fig. 1) [ 18]. This procedure will certainly gain recognition as the importance of the orientation of the immobilization of protein ligands becomes more obvious. Several examples of the necessity for a proper ligand immobilization have been reported recently. One of the most interesting is the development of an oriented immobilization of protein A as an IgG alfinity matrix [19o]. The use of immobilized protein A as the coupling agent and spacer for immobilizing the antibody yields an immunoabsorbant with twice the capacity of that achieved using cyanogen bromideactivated agarose. The presumption is that protein A orients the IgG molecules by binding to their Fc portions. This forces the antigen-binding portion of the molecule into the solution, thereby increasing its binding capacity. Similarly, hydrazide agarose and cellulose can be used to
Affinity chromatography for protein isolation Scouten 39
I Cleavablearm I II
Matrix~ J ~
U
~
Attachment point
I Po'nt
Reporter~ P r o t e i n
-9,oetector I
I moiety ]
I
I-'.Pr°,te'"
lattpCnntentl
n Matrix]
I
+N
roteio I
orient periodate, or enzyme-oxidized IgG [20 o.,21.,22.. ], to increase the antigen-binding capacity of the matrix (Fig. 2).
Fig. 1. A schematicrepresentationof an immobilized protein modification reagent. Reprinted with permission from [18].
in another demonstration of the significance of the mode of ligand immobilization, Gabius [23] showed that the type of both linkage and spacer arm used to immobilize carbohydrate ligands made a significant difference to their effectiveness as an aWmitychromatography material for the purification of ~)-galactoside-binding proteins. In a similar study, three ubiquitin carboxyt-terminal hydrolases were affinity-purified by ubiquitin immobilized to a matrix via its arginine residues. In contrast, these same carboxyl terminal hydrolases were unable to recognize ubiquitin immobilized via its b/sine residues [24].
Chromatographic matrices
~Fab//'~~Fab
////v
Fab
Fig.2. Schematicdepictingthe inferreddifferencesin the orientationof antibodies immobilizedvia orientedoligosaccharidespecificchemistriesand randomamino-acid-directedchemistnes.Reprintedwith permissionfrom [21o].
One of the most interesting and novel of the chromatographic systems reported in the past year is the development of immobilized artificial membrane chromatographic materials [25"]. These matrices consist of a silica support to which a long-chain fatty add has been coupled. Normally this is done using a long dicarboxylic acid which is coupled to an amino silica surface at one end via an amide bond. Subsequently, the remaining free carboxyt is coupled to a phosphatidyt lipid to create a surface that mimics a cell membrane. Membrane proteins bind to, and are easily chromatographed on, the resulting artificial membrane surface. As membrane proteins have been notoriously difficult to handle, this material should prove to be very useful in their isolation and study. Investigation of thiophih'c absorbants as inexpensive replacements for protein A chromatography materials for IgG purification has continued; although they have not yet become wholesale replacements for protein A, it seems likely that they will do so in the near future. These resins
40 Analytical biotechnology are prepared by reacting an amino matrix with an appropriate reactive reagent containing a thiol group. For example, amino silica can be reacted with divinyl sulphone which, in turn, is treated with a large excess of mercapto-ethanol. Such a column is very efficient at purifying IgG and, for all intents and purposes, has practically the same selectivity for IgG as protein A, but without the expense and biological instability that accompanies the latter [26"]. A similar small ligand replacement medium for protein A agarose has been developed by Ngo and co-workers [27] based on pyridinyl derivatives.
Applications One of the most striking applications of affinity chromatography in the past year has been the purification of human erythrocyte acyl phosphatase [28.]. Three hundred milliliters of erythrocytes were used to purify 330 pg of the enzyme using an initial affinity chromatography step. This step, based on immunoaffinity chromatography on anti-acyl phosphatase antibodies immobilized to Sepharose 413, effected a 135 000-fold purification in a single step with a 44% yield. Subsequently, the enzyme was purified to homogeneity by gel filtration combined with reversed-phase chromatography for a total 255 000-fold purification. The immunoaffmity step yielded the greatest purification of the three steps and it can be speculated that a second irnmunoal~ty step might have created an homogeneous enzyme as easily as (or easier than) the non-affinity procedures. Another striking example of affinity chromatography was the large scale purification of vasparaginase, an enzyme with significant anti-neoplastic activity, from E r w i n i a carotovora [29"]. The enzyme was purified from 880 liters of whole fermentation broth using a three-step process. Treatment with acetone reduced the volume of the broth and subsequent chromatography on L-aspa-
ragine immobilized on Sepharose 6-Fast Flow produced an 8.5-fold purification with a recovery of 38% of the enzyme. Dialysis and further concentration of the enzyme increased its purification to 8.9-fold with a continued 38% recovery. Although the degree of purification in this case may seem insignificant compared with the 255 O00-fold obtained by the affinity step in the purification of erythrocyte acyl phosphatase, the cell-free fermentation broth that was used as the source of enzyme contained relatively small quantities of any type of contaminant. The most striking thing about this process is the scale at which the purification can be easily carried out. The L-asparagine Sepharose 6-Fast Flow column measured 25.2cm by 18cm and contained a volume of 9 liters. The cell-free extract consisted of 1400 liters of fermentation broth clarified through an inline filtration device and loaded in a reversed flow fashion in order to prevent contamination of the column during the process. Flow rate was a significant 60 liters per hour and 10-liter fractions were collected. Under such conditions, the majority of the enzyme was eluted in a single enzyme-rich fraction and 33.6 g of active protein was obtained.
Protein quantification and visualization Quantification is extremely important in any protein purification process as well as in the preparation of affinity chromatography materials that involve immobilized proteins. Gaur and Gupta [30 °-] have developed a very successful method for the estimation of amino groups on polymer supports based on the high extinction coefficient of the trityl moiety (Fig. 3). Initially, protein is trityiated using either sodium dimethoxytritytbutamte or 2-4-dinitrophenyl dimethoxytrityibutarate. The tritylation process can be controlled so that only amine functions on the polymer support are tritylated. When the excess reagents have been removed, the dimethoxytrityl cation can be released by treatment with perchloric acid. The
Fig. 3. Reactions involved in the spectrophotometric determination of solidsupported amino groups with ~'rnax= 498nm and E49a=70000M-1cm -1. DDTB, 2-4-dinitrophenyl dimethoxytritylbutarate; DMAP, dimethyl aminopyridine; DMF, dimethyl formamide; P, polymer support; SDTB, sodium dimethoxytritylbutarate. Reprinted with permission from [29".].
Affinity chromatography for protein isolation Scouten absorbance of the released cation, which has an extinction coefficient at 498 nm of 70 000 M - l c m - 1, is then measured spectrophotometrically. The high extinction coefficient of the trityl cation allows the very sensitive determination of amines on the polymer matrix. This is of obvious value in quantifying proteins immobilized to an affinity support material. Conversely, the method can be used to determine the number of amine groups on an amine-modified matrix, which is to be used for the immobilization of an affinity ligand. Another approach to protein quantification and visualization that has been exploited over the past year and is expected to become far more widely used in the future, involves recombinant fusion proteins in which protein A has been fused to an enzyme that can convert a colorless soluble substrate into a highly absorbing insoluble product. Such fusion proteins are especiaUyuseful in visualizing proteins on membranes or electrophoretic gels. The procedure involves electrophoresis of the sample followed by incubation with an antibody against the protein(s) being visualized or quantiffed. The gels are then washed and incubated with the fused enzyme-protein A. The gel is, subsequently, washed and the substrate mixture is added. After an appropriate incubation time, the protein desired (and only that protein) appears very distinctly as a colored band on the gel. This method has been demonstrated to be suitable for the quantitative deternlination of samples as small as 100 pg when protein A-neomycin phosphotransferase fusion protein is used [31o]. Similarly, the fusion protein of metapyrocatechase-protein A [32 o] has permitted the detection of bovine serum albumin in the concentration range of 0.1 n g m l - 1 to I m g m l - 1.
recent advances in biotechnology. The procedures described here, particularly temperature-programmed elution, centrifugal affinity chromatography, and the coupling of genetic engineering with affinity chromatography as 'preplanned purification' or 'affinity handles', represent major advances in the continuing development of the field. Moreover, the coming year is likely to bring as many, if not more, significant advances. Ultimately, each of these will be useful in valuable commercial and clinical applications of solid-phase biochemistry.
References and recommended reading Papers of special interest, published within the annual period of review, have been highlighted as: • of interest oo of outstanding interest 1. oo
WU.CHEKM: 8th International Symposium on Affinity Chromatography and Biological Recognition. J OJromatogr 1990, 510:1-2. This is the lead article for the symposium issue related to the 8th International Affinity Chromatography Conference. This volume is a conpendium of the most recent advances in affinity chromatography and similar purification methods. 2.
SCOUTENWH (ed): AffiniOJ Chromatography 1981, New York: Wiley Interscience 59:1-338.
3. •
CLAUSENH, WHITE T, TAKIO K, TrrANI K, STROUD M, HOLMES E, KARKOVJ, Tram L, HAKOMORI S: Isolation o f Homogeneity and Partial Characterization of a Histo-Blood Group A Defined Fuc~tl --*2Galctl ---,3-N-Acetylgalactosaminyltransferase from Human Lung Tissue. J Bid Chem 1990, 265(2):1139-1145. This is an excellent, but not representative, example of m o d e m applications of affinity chromatography to the purification of proteins present in trace amounts in a given sample. 4.
STEERSE JR, CUATRECASASP: [3-Galactosidase. Methods gnzymol 1974, 34:350-358.
Affinity handles
5.
WEISSBACHA, POONIAN M: Nucleic Acids Attached to Solid Matrices. Methods Enzymol 1974, 34:463-475.
Downstream processing has often been the major bottleneck in the industrial production of proteins. However, during the last 5 years, a novel protein purification technique has been developed that is based on gene-fusion technology. A DNA fragment encoding an additional potypeptide fragment is fused to either the 5' or 3' end of the gene of interest (for an excellent review see [33]). The properties of the additional polypeptide tail can be taken advantage of during purification of the obtained protein product. A number of genes specifically modified to simplify protein purification have been described. Recently, such 'affinity handles' have been used to facilitate hydroprobic [34], covalent [35] and metal chelate separations [36].
6.
GRENOTC, CUILLERONCY: Isolation of Antibodies to Steroid Hormones by Affinity Chromatography on Antigen-Linked Sepharose: an Efficient Electrophoretic Elution Procedure. Bioclx,m Biophys Res Commun 1977, 79:274-279.
Conclusion Affinity chromatography and the related aspects of solidphase biochemistry have become significant elements in
7. •.
BERGOLDAF, CARR PW: Improved Resolution of Glycoproteins by Chromatography with Concanavalin A Immobilized on Microparticulate Silica via Temperature Programmed Elution. Anal Chem 1989, 61:1117-1128. This is an excellent description of an extremely novel technique for the elution of proteins from lectin columns using a temperature-programmed elution method. This should be applicable to a wide variety of affinity chromatographic systems, other than lectin/glycoprotein systems, and may well find its place in many future investigations. 8. BERG A, ScotrmN WH: Dye-Ligand Centrifugal Affinity Chro•. matography. Bioseparation 1990, 1:23-31. This describes a method for screening many different types of columns using a single sample type. In this sense, it is complementary to, and the reverse of, HPLAC. In the article, various dyes are screened for their ability to bind the Fc fragment of IgG. Such dyes could be of considerable importance in antibody purification, immobilization and application in enzyme-linked immunosorbent assays. 9. ,
HONDMANNDHA, VlSSERJ: Screening Method for Large Numbers of Dye-Absorbents for Enzyme Purification. J t2bro~ matogr 1990, 510:155-164.
41
42
Analytical biotechnology This paper presents a rapid method for screening affinity ligands using microtiter plates. Many dye-ligands can be screened for their ability to bind protein rapidly. The system is particularly applicable to enzymes with chromophoric substrates. PRICKETTKS, AMBERGDC, HOPP TP: A Calcinm-Dependent Antibody for Identification and Purification of Recombinant Proteins. BtoTechniques 1989, 7:580-589. This is an introduction to a metal ionM_ependent immunosorbant that can be used for the purification of recombinant proteins that do not yield readily to classic purification methods.
rain to hydrazide cellulose. The technique is also demonstrated to be extremely useful in the preparation of an absorbant for concanavalin A and similar lectins. 23.
GAmUSH-J: Influence of Type of Linkage and Spacer on the Interaction of [~-Galactoside-Binding Proteins with Immobilized Affinity Ligands. Aria/B/ochem 1990, 189:91--94.
24.
DUERKSEN-HUGHESPJ, WILLIAMSONMM, WILKINSON KD: Affinity Chromatography Using Protein Immobilized via Arginine Residues: Purification of Ubiquitin Carboxyi-Terminal Hydrolases. B/oc.bem/ary 1989, 28:8530--8536.
10. ..
11.
HoPP TP, PRICKETr KS, PRICEVL, LIBBYRT, MARCHCJ, CERRETrI DP, UROALDL, CONLON PJ: A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification. B ~ . b n o / o g y 1988, 6:1204-1210.
12.
SASSENFELDHM: Engineering proteins for purification. Trends Bk:~chno/1990, 8:88-93.
13.
I~GRICESFJ, GRUNINGER-LErlX=HF: Rapid Purification of Homodimer and Heterodimer HIV-1 Reverse Transcriptase by Metal Chelate AfBnity Chromatography. Eur J Biochem 1990, 187:307--314.
14. •
REARDONJE: Herpes Simplex Virus Type 1 DNA Polymerase: Mechanism-Based AtFmiW Chromatography. J B~/ OJem 1990, 265:7112-7115. This paper describes a novel method of p r o m elution by means of 'mechanism-based' affinity chromatography. The technique will be very useful in those cases where mandatory sequential substrate binding kinetics exist and where appropriate substrates and inhibitors can be obtained. It may not be widely applicable buL where it can be employed, it will be extremely effect~e. 15.
NARAYANANSP,, CRANELJ: Affinity Chromatography Supports: a Look at Performance Requirements. Trends Biotechnol 1990, 8:12-16.
16.
SCOUTENWH: A Survey of Enzyme Coupling Techniques. Methods Enzymol 1987, 135:30-65.
17.
MARBURGS, FIANAGANME, TOLMANRI, RICHARDI~ Chemistry of Solid Supports: Defining Events and Titers by Use of Cleavable, As,sayable Linking Molecules. Anal Biochem 1989, 181:242-249.
18.
LEWISC, SCOtrlEN WH: Immobilized Protein Modification Reagents. In SO//,/Phase B ~ / s t r y edited by Scouten WH. New York: Wiley interscience, 1983, pp 665-678.
19. •
AVIIADM, KAUSHALV, BARNESLD: Immunoaffmity Chromatography of Diadenosine 5'5"-PlJ~4-Tetraphosphate Phosphorylase from Saccbaromyces cerevtstae. Biotechnol Appl
B/oc/aem 1990, 12:276-283. This is an excellent example of the oriented immobilization of antibodies producing a significantly enhanced capacity when contrasted to classically prepared immunosorbams.
25. .
MARKOWCHRJ, STEVENSJM, PIDGEON C: Fourier Transform Infrared Assay of Membrane Lipids Immobilized to Silica: Leaching and Stability of Immobilized Artificial MembraneBonded Phases. Aria/Bt~bem 1989, 182:237-244. This is the latest in a series of papers describing the preparation, application, and characterization of immobilized artificial membrane afBnity matrices. Such matrices appear to be superior in the isolation of membrane-bound proteins. 26. •
NOPPERB, KOHEN F, WILCHEK M: A Thiophilic Adsorbeflt for the One-Step High-Performance Liquid Chromatography Purification of Monoclonal Antibodies Anal Biocbem 1989, 180:66-71. This papers describes an excellent recent application of thiophilic adsorbents for the purification of antibodies. This method may well replace protein A chromatography. 27.
NGO Tr, KHATFERN: Chemistry and Preparation of Affinity Liganda Useful in Immunoglobulin Isolation and Serum Protein Separation. J Oyroma~gr 1990, 510:281-291.
28. .
DEGL'INNOCENTID, BImTI A, STEFANIM, LtGUR1G, RAMPONIG: Immunoamnity Purification and Immunoassay Determination for Human Erythrocyte Acylphosphatase. B~,,c,b ~ p l BR~c/'~m 1990, 12:450-459. A report of an exceptional application of afBnity chromatography for the purification of an enzyme present in trace quantities. 29.
LEE S-M, WROBLEMH, ROSSJT: L-Asparaginase from E n v t n t a carotovorat an Improved Recovery and Purification Process Using Affinity Chromatography. ~ B/ocbem BR~tecb no/1989, 22:1-11. L-Asparaginase is purified from 880 liters of fermentation broth. This is an excellent presentation of large scale enzyme purification by atBuity chromatography for eventual biomedical and therapeutic application. •.
30. ••
GAURRig, GUPTAKC: A Spectrophotometric Method for the Estimation of Amino Groups on Polymer Supports. Anal B/ocbem 1989, 180:253-258. A very sensitive method for determination of the amine groups on immobilized matrices has been needed for a long time. This paper reports such a method.
20. •.
HUNGER H-D, SCHMIITr G, FIACHMEIER C, BEHRENDT G, COUTELLEC: High-Sensitivity Protein Detection by a New 'Contact-Copy' Method Using a Protein A-Neomycin Phosphotransferase II Fusion Protein. Ana/ B/c~bem 1990, 186:159-164. This is one of many recombinant protein A-enzyme fusion proteins being used for the visualization and quantification of antibodies and/or proteins recognized by antibodies. In general, the method aliows very sensitive quantification and visualization of specific proteins and should prove to be exceptionally useful in future investigations.
21.
32. •
22. **
33.
DOMENPL, NEVENSJR, MAIJ~ AK, HERMANSONGT, KI.ENKDC: Site-Directed Immobilization of Protein. J Cbroma~gr 1990, 510:293-302. This paper compares several commercial activated affinity chromatographic matrices with regard to their ability to immobilizeIgG and prepare affective immunosorbant matrices. O'S~NNESSYDJ: Review: Hydrazido-Derivatized Supports in Affinity Chromatography. J GM~matogr 1990, 510:13-21. ~ns' is a comprehensive review of the use of hydmzides for the immobilization of giycoproteins and other aldehyde-containing, or potentially aldehyde-containin~ molecules. TLmKOVAJ, PETKOV I~ SAJE~KJ, KAS J, BENES MJ: Carbohydrates as a Tool for Oriented Immobilization of Antigens and Antibodies. J ~ 1990, 500:585-593. This article details the preparation of a biospecific immunosorbant for ovalbumin antibodies prepared by coupling period,ate-oxidized ovalbu-
31. •
KOBATAKEE, NISHIMOmY, IKARIYAMAY, AIZAWAM, KATO S: Appfication of a Fusion Protein, Metapyrocatechase/Protein A, to an Enzyme Immunoassay. Anal BiocT~om 1990, 186:14-18. Like [31.], this paper describes an application of a protein A-enzyme fusion protein, but using a different chromophore and enzyme system for color development. SASSENFELDHM: Engineering Proteins for Purification. Trends BiotecJTno/1990, 8:88-93.
34.
PERSSON M, BERGSTRAND M, B~LOW L, MOSBACH K: Enzyme Purification by Genetically Attached Polycysteine
Affinity chromatography for protein isolation Scouten
35.
Loat~ G, PERSSONM, BOLOWI, MOSBACHI~ Metal Affinity Prccipitation of Protei~ Carryin~ Genetically Attached Polyhistidine Affinity Tails. Eur J Biochera~ 1991, in press.
and Polyphenylalanine Affinity Tails. Anal Biochem 1988, 172:3~0--337.
36.
PERSSON M, BOLOW L, MOSBACH K: Purification and Sitespecific Immobilization of Genetically Engineered Glucose Dehydrogenase on Thiopropyl-Sepharose. FEBS Lett 1990, 270:41-44.
WH Scouten, Department of Chemistry, Bayior University, Waco, Texas 76798, US&
43
