Avidin Column as a Highly Efficient and Stable Alternative for Immobilization of Ligands for Affinity Chromatography Edward A. Bayer and Meir Wilchek* Department of Biophysics, The Weizmann Institute of Science, Rehovot 76100, Israel
The avidin/biotin system was applied as a general mediator in the adsorption/desorption or immobilization of biologically active macromolecules to solid supports. In this context, model biotinylated proteins (lectins and antibodies) were attached to avidin-coupled Sepharose. As examples for affinity chromatography, peanut agglutinin and anti-bansferrin antibody were used to isolate asialofetuin and transferrin, respectively. The capacity and product yields were significantly better than those achieved with conventional affinity chromatography on CNBractivated Sepharose columns containing the same lectin or antibody. Moreover, the columns were characterized by improved stability properties exhibiting remarkably low levels of leakage.
INTRODUCTION Over the past two decades, the general trend for the purification of biologically active compounds has clearly favored the use of affinity chromatography (Wilchek et al., 1984). Using this technology, one usually binds a ligand chemically to a carrier matrix; the immobilized ligand then recognizes a biological target molecule to be purified. This approach is in many ways very appealing, but in laboratories where more than one product is routinely purified, many different types of affinity columns must be prepared and maintained. One of the difficult steps in preparing an affinity column is the chemical activation of the inert matrix prior to attachment of the biologically active ligand. Historically, cyanogen bromide activation of Sepharose was first employed for this purpose (Cuatrecasas et al., 1968). The original procedure is still widely in use today, despite the hazards involved in CNBr activation and innovative development of alternative activating reagents (Wilchek and Miron; 1982, 1985; Kohn and Wilchek, 1984). Moreover, the resultant isourea bond is unstable, thus causing leakage of the affinity ligand and consequent reduction in capacity and specificity (Wilchek et al., 1975; Kohn and Wilchek, 1982). In addition, storage of activated matrices consistently results in reduced levels of coupling compared to that of the freshly prepared affinity column. Many biochemists are uncomfortable with chemical reactions. It would therefore be convenient to have an activated carrier to which the ligand can be coupled Abbreviations used: Ab, anti-human transferrin antibody; B, biotinyl moiety: B-cap.but, biotinyl Nc-aminocaproyl-W-aminobutyryl moiety; BNHS, biotinyl N-hydroxysuccinimide ester; BHZ, biotin hydrazide; PBS, 10 m M phosphate buffered saline (pH 7.4); PNA, peanut agglutinin; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. * Author to whom correspondence should be addressed.
biologically. An avidin-containing column is appropriate for these purposes (Bayer and Wilchek, 1978; Wilchek and Bayer, 1989). Alternatively, an antihapten antibody column with an association constant greater than lo8 M would also be suitable (Wilchek and Gorecki, 1973). In this context we have previously promoted the use of avidin/biotin technology for improving the versatility of affinity chromatography as a method in general (Bayer and Wilchek, 1980; Wilchek and Bayer, 1983; 1984). Such an ‘activated’ column would contain either egg white avidin or bacterial streptavidin to which biotinylated ligands can then be bound with exceptionally high stability. As we have suggested in the past (Wilchek and Bayer, 1988), virtually any ligand, including antibodies, binding proteins, enzymes, cofactors, inhibitors, drugs, hormones, nucleic acids, etc, can be easily biotinylated and bound to an avidin column. Many biotinylated biomolecules are commercially available. Such immobilized ligands would then be used for the purification of an appropriate molecular counterpart. A distinct advantage of this system would be that following preparation of the initial avidin-containing affinity matrix, additional chemical manipulations would not be required. Indeed, such an approach was used for the analytical isolation of cell surface antigens (Updyke and Nicolson, 1984) and lectins (Buckie and Cook, 1986). However, the system can also be exploited for the immobilization of precious biotinylated ligands which are available in very limited quantities as well as for the large scale preparation of desired biomolecules (e.g., antigens using monoclonal antibodies). In this study, we describe conditions for efficient immobilization of biotinylated lectins and antibodies to an avidin/Sepharose column. The resultant affinity columns were used to demonstrate a model for large scale purification of glycoproteins and antigens. Interestingly, the performance of these columns was far superior to that of the same affinity system prepared by conventional methodology, as reflected by the respective capacities, yields and lack of leakage.
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and containing about 1.6 mg transferrin per mL Sepharose) was washed sequentially with PBS, 0.2 M acetic acid and again with PBS. Rabbit anti-human transferrin antiserum (7 mL dissolved in an equivalent volume of PBS) was applied to the column, and the column was washed with PBS until the effluent fractions registered optical densities (280 nm) of less than 0.03. The eluant solution (0.2 M acetic acid) was then applied and the column was washed until the optical density dropped below 0.05. The fractions (65 mL total) were pooled, dialysed against PBS, centrifuged to remove precipitates, and concentrated in an Amicon ultrafiltration unit (Lexington, USA). The preparation was stored in 0.5 mL aliquots at a concentration of 2.3 mg/mL, total yield 13.7 mg.
EXPERIMENTAL Materials. The following materials were obtained from Sigma, St. Louis, MO, USA: biotin, iminobiotin, asialofetuin (type I ) fetuin (type IV), Coomassie brilliant blue R, and a high molecular weight standard kit containing the following markers: myosin (205 000), P-galactosidase (1 16 000), phosphorylase b (97 400), bovine serum albumin (BSA) (67 000), ovalbumin (45 000), and carbonic anhydrase (29 000). Egg white avidin was generously provided by S. C. Belovo (Bastogne, Belgium). Peanut agglutinin (PNA) was a generous gift by Professor Nathan Sharon of our department. Rabbit anti-human transferrin antiserum was a generous gift by Dr Harry Langbeheim of BioMakor, Inc. (Rehovot, Israel). Human plasma was obtained from the Magen David Adom Blood Bank (Jaffa, Israel). Preparation of avidin/Sepharose column. In the experiments described in this work avidin/Sepharose was prepared by the conventional CNBr-activation procedure. In later experiments, equivalent (and sometimes improved) results were obtained using either the 'cyano-transfer' activation procedure (Kohn and Wilchek, 1984) or chloroformate-activation of Sepharose (Wilchek and Miron, 1982). For stability considerations, the latter procedure is the preferred method for column preparation. The avidin columns can be stored at 4 "C for at least 5 years in the presence of 0.1 Yo sodium azide and can be used immediately after a short wash protocol using successive passages of equilibration and elution buffers. Preparation of biotinylated peanut agglutinin (B-PNA). In this work, PNA was used as a model binding protein. In initial studies, we were interested in calibrating the correlation between the number of biotin molecules attached to a representative binding protein and its subsequent attachment to an avidin column. For this purpose, varying molar ratios of biotinyl N hydroxysuccinimide ester (BNHS) were used to biotinylated peanut agglutinin (B-PNA). PNA, at a concentration of about 2.5 mg/mL was dissolved in a 0.1 M NaHC03 solution containing 0.2 M NaCI. The solution was centrifuged at 1500 x g for 10 min at 25 "C to remove precipitates. The supernatant fluids were collected and the optical density (280 nm) was measured. The solution was adjusted to 20 p~ (EL: = 7.7) protein. A 20 mM dimethyl formamide solution containing BNHS (6.8 mg/mL) was prepared, and 0,20,40,80 and 200 pL, respectively, were added to 4 mL samples of the prepared PNA solution (80 nmol PNA per sample). The final dimethyl formamide concentration of each sample was adjusted to 5%. Each reaction mixture was allowed to stand for 2 h at 25 "C, and each sample was dialysed separately, first against 0.15 M NaCl and then against PBS. The samples were collected and in each case about 90% of the initial protein could be recovered. The preparations were stored at -20 "C in 1 mL aliquots. Affinity isolation of anti-transferrin antibodies. A column (20 mL resin) containing Sepharose/transferrin (prepared by the conventional CNBr-activating procedure
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Preparation of biotinylated anti-transferrin antibodies. I , B-Ab. A solution of anti-transferrin antibody (3.4 mg in 1.5 mL) was treated with 50 pL BNHS solution (4 mg/mL in dimethyl formamide). The reaction was allowed to proceed for 2 h at 25 "C, and the reaction mixture was dialysed exhaustively against PBS. 2, Bcap.but-Ab. A dimethyl formamide solution (30 pL) of B-cap.but-NHS (10 mg/mL) was added to antitransferrin antibody (3.4 mg in 1.5 mL). After 2 h at 25 "C, the reaction mixture was dialysed exhaustively against PBS. 3, BHZ-Ab. Biotinylation of antitransferrin antibody was carried out by a slight modification of the procedure described by O'Shannessy et al. (1984). Antibody solution (3.4 mg in 1.5 mL PBS) was oxidized with 10 mM (final concentration) sodium periodate. The solution was dialysed for 4 h against PBS and 4 mg (solid) BHZ was added. After a 2 h incubation period at 25 "C, the reaction mixture was dialysed exhaustively against PBS. Preparation of affinity columns. Samples of the biotinylated
binding protein (lectin or antibody) were applied to the avidin column. The absorbance (Azxo)of the applied and effluent fractions were examined in order to estimate the extent of binding. We usually apply between 0.5- to 2-fold molar ratios of the biotinylated protein to avidin. One approach to establish the preferred ratio of the column constituents is to determine the number of free biotin binding sites before and after application of the biotinylated protein to the column. This can be accomplished by several methods, e.g., spectrophotometrically (Green, 1970), fluorometrically (Mock et ul., 1988) or using radioactive biotin obtained from either Amersham (Amersham, UK) or New England Nuclear (Dreiech, FRG). A second approach is to apply successive amounts of the biotinylated protein and to determine applied vs effluent readings of each application. In this case, initial applications of the biotinylated protein are usually adsorbed entirely by the avidin column; subsequent applications are only partially adsorbed. This approach gives an empirical indication of the capacity of the avidin column for a given biotinylated protein. It is usually advisable not to overload the column; a reasonable measure for an effective affinity column is to leave about two thirds of the original number of biotin binding sites or to load about one third of the column capacity with the desired biotinylated protein.
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Quantitative affinity chromatography. A small (0.5-1 .O mL) affinity column containing the desired resin was used in each case. 1 mL fractions were collected manually using a 1 mL volumetric flask. The amount of protein per fraction was usually determined spectrophotometrically, and the purity of the fractions was, in some cases, confirmed either by SDS-PAGE or by an appropriate dot-blot assay. In the case of the PNA columns, the adsorbed asialofetuin was released using a PBS solution containing 0.5 M galactose. In the case of the antibody column, a solution of 0.1 M acetic acid was used to release the antigen.
Gel electrophoresis. Polyacrylamide gel electrophoresis was
performed using 10% separating gels in the presence of sodium dodecyl sulfate (SDS-PAGE) under conditions described previously (Bayer et al., 1987).
RESULTS Biotinylated lectins In order to determine the feasibility of such systems, we first examined the immobilization characteristics of a simple purified binding protein for isolating a purified macromolecule. As a model system for this study, peanut agglutinin (PNA) was biotinylated, the biotinylated lectin (B-PNA) was bound to avidin-coupled Sepharose, and the product was used for the isolation of a commercial preparation of a galactose-containing glycoprotein (asialofetuin). PNA was subjected to biotinylation by conventional procedures using various molar ratios (relative to the concentration of protein) of biotinyl-N-hydroxysuccinimide ester (BNHS). Optimal binding to Sepharose/avidin affinity columns was achieved at an initial BNHS:PNA ratio of about 10: 1 (Fig. 1). The exact ratio of biotin moieties (average) per protein molecule was not determined in these experiments. In our experience, such an assessment is mainly of academic value. It is more relevant to gauge the interaction of a biotinylated preparation with an avidin probe. Again, experience has taught us that the interaction with an immobilized form of avidin is usually preferred, since the value obtained gives an indication of the percent of molecules in the preparation which will interact with the avidin-containing probe. On columns containing 1 mg avidin per mL Sepharose, 3.5 mg B-PNA could be readily attached. The capacity and elution efficiency of such columns for asialofetuin was much greater than those of conventional PNA-containing columns which were prepared using the standard CNBr-activation method. Samples of asialofetuin were loaded onto the two types of affinity column. In the case of the conventional PNA/Sepharose column ( I .7 mg protein per mL resin), the capacity was quite low; only 0.37 mg asialofetuin could be loaded onto 1 mL of the column. A more 104 JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 3 , 1 9 9 0
100
=0
75
c
c
a, t
50 8
25
r :7 A
Figure 1. Interaction of biotinylated PNA preparations with avidin/ Sepharose column. Purified samples of PNA were biotinylated using various molar ratios of BNHS. Following dialysis, the respective biotinylated preparations were applied to an avidin column and the percent retained (percent protein applied minus percent in effluent) was determined.
serious problem, however, was the finding that only 0.24 mg of the glycoprotein could be selectively released from the column using 0.5 M galactose, indicating that about a third of the target material was very tightly bound. Thus, conventional PNA/Sepharose is a relatively ineffective affinity column. On the other hand, the performance of the PNA column prepared using avidin/biotin technology was excellent. The capacity of the column was comparatively high (1.2 mg per mL column) and the entire amount could be released selectively using the competing sugar. The eluted protein appeared pure by SDS-PAGE and by selective labeling of the blotted sample (data not shown). The product was not contaminated by detectable levels of either B-PNA or avidin. The results suggest that more effective affinity columns may be achieved through aviding/biotin mediation. Attempts were made to release the B-PNA from the Sepharose/avidin column, such that the latter would be reusable. The method comprised elution of the biotinylated lectin using solutions of iminobiotin at pH 1 1. About 70% of the biotinylated lectin could be recovered in this manner.
Biotinylated antibodies The results described above for biotinylated lectins were extended for application to biotinylated antibodies. Commercially prepared (BioMakor, Rehovot, Israel) polyclonal antibodies, elicited against human transferrin, were used as a model system. The characteristics of three different biotinylating reagents were compared for effectiveness in incorporating biotin into the antibody molecule. These were: 1, BNHS; 2, a longer chained derivative, biotinyl NC-aminocaproyl-Nuaminobutyryl-N-hydroxy-succinimideester B-cap.but NHS); and 3, biotin hydrazide (BHZ). The first two labels were used to biotinylate amino groups of the antibodies and the hydrazide derivative was used to label the sugar residues of the periodate oxidized antibody.
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The capacity of the avidin column for the antibody preparations biotinylated via amino groups was greater than that observed for the BHZ-derivatized antibody (Table 1 ). A typical column contained 1.5 mg avidin per mL Sepharose, and about 1.3 mg/mL B-Ab. All three types of the columns could be used to isolate transferrin (Table 2); the capacity of the columns was about 0.4 mg transferrin per mL Sepharose. A new column was prepared which contained 2 mg B-Ab per 1 mL Sepharose/avidin. Human plasma (diluted fourfold with PBS) was applied directly to this column. After extensive washing of this column, the adsorbed transferrin was released using 0.1 M acetic acid. Eluant fractions were collected, and the column was regenerated by washing with PBS. A second sample of serum was applied and eluted in an identical fashion. Absorbance measurements of the eluted fractions indicated that about 0.9 mg was purified initially and another 0.75 mg sample was purified upon regeneration of the column. Subsequent trials showed a stabilization of the column, and about 0.7 mg per mL column was consistently isolated per cycle. Both preparations were equally pure by SDS-PAGE (Fig. 2). Only very low levels of other contaminating serum proteins could be detected. The purified transferrin appeared to contain even less contaminating antibody than that of the commercially available (Sigma) protein (against which the
Table 1 . Binding of biotinylated anti-transferrin preparations to avidin/Sepharose B~Ab
Applied Effluent Bound initially AcOH wash Total bound
0.85 0.02 0.83 0.15 0.68
6-cap.but-Ab
0.67 0.01 0.66 0.06 0.60
BHZ~Ab
0.75 0.31 0.44 0.21 0.23
Values represent amount (mg) of the designated biotinylated antibody preparation which was present in each fraction. Column volumes of 0 5 mL were used in each case.
Table 2. Isolation of commercial preparation of transferrin on biotinylated antibody bound to avidin/Sepharose 6-Ab
6-cap but-Ab
BHZ Ab
0.50 0.16 0 34 0.20
0.50 0.27 0.23 0.18
0.50 0.36 0.14 0.15
0.45 0.13 0.28 0.17
0.47 0.22 0.25 0.22
NT
Trial 1
Applied Effluent Bound AcOH eluant Trial 2
Applied Eff Iuent Bound AcOH eluant
~
-
Values represent the amount (mg) of transferrin contained in each fraction. Column volumes of 0.5 mL were used in each case. NT, not tested.
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A
B
C
D
Figure 2. SDS-PAGE of transferrin purified directly from human plasma on a B-Ab column. Lane A shows the pattern of human plasma, a sample of which was applied to the column; lane B shows the pattern of transferrin purified on the B-Ab/avidin/Sepharose affinity column; lane C gives the pattern of Sigma transferrin; and lane D shows the position of molecular weight protein standards (205 000, 116 000, 97 400, 67 000, 45 000 and 29 OOO), Samples of 30 pL were added to each well.
anti-transferrin antibodies used in this study were prepared).
DISCUSSION We have shown that avidin columns can be used to bind different biotinylated proteins and that the resultant affinity column is appropriate for the selective isolation of biologically active target molecules from crude material. The biotinylation step is very easy to perform, and most biotinylated proteins can be stored in the active state indefinitely. In this sense, the avidin column is a ‘preactivated’ column, and the biotinylated protein can be attached within minutes. In applying this approach, we have demonstrated the use of immobilized proteins for the isolation of their biological counterparts, e.g., biotinylated antitransferrin for transferrin and biotinylated PNA for asialofetuin. In each case, the target protein could be eluted from the column, and the affinity column could be reused. The isolated protein was remarkably pure as tested by SDS-PAGE. The columns proved to be more efficient than standard affinity chromatographic techniques (on conventional columns). Using a polyclonal antibody preparation against human transferrin, we examined three different biotinylation procedures to determine their effect on JOURNAL OF MOLECULAR
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purification of the model antigen. I t was clear that higher levels of biotinylation could be obtained by biotinylating via the amino groups o f the antibody compared to that achieved through periodate oxidation of the sugars. Using the latter procedure (coupling of biotin hydrazide to periodate-generated sugar aldehydes o f the antibody), about half of the amount of biotinylated antibody which was initially bound to the column was released upon washing with acetic acid. Immobilization achieved through biotinylation via the peptide linkage was more stable (see Table 2). The best results were achieved using a long chain reagent bound to the amino groups of the antibody. In the case of B-cap.but-Ab, the stability of the resultant avidinjsepharose affinity column to the acetic acid washing step was greater than that of the shorter chained B-Ah. In this work, however, we did not attempt to couple a long chain biotin-containing derivative (e.g., biocytin hydrazide) through the sugar residue of the antibody; consequently, we cannot state conclusively whether the reduced levels of binding observed with the biocytin hydrazide conjugate is solely related to the chemistry of biotinylation via the sugar or whether a long chain derivative of biocytin hydrazide would have improved the stability characteristics or capacity of the column. In any event, it should be noted that all three of the resultant immunoaffinity columns could be used successfully to isolate the antigen in question. Once the biotinylated binding protein is applied to the avidin column and subjected to the stringent washing procedure, the affinity column is remarkably stable. In this regard, it is advised to apply subsaturating amounts of the biotinylated protein such that a population of residual free biotin binding sites will remain. These would then be available to crosslink biotinylated protein which may disengage from the avidin matrix due to the low but finite dissociation constant. More recent work in our laboratory has shown that avidin coupled to Sepharose using the chloroformate activation procedure is preferred to that coupled via CNBr. Nevertheless, even using CNBr-induced coupling of avidin, the resultant columns were remarkably stable compared to conventional antibody columns prepared by direct CNBr linkage. The addition of the biotinylated protein thus seems to improve the leakage characteristics
of the column, perhaps due to crosslinking among the avidin molecules. We noted also that the capacity of the columns was somewhat reduced after the first trial, reaching a relatively stable plateau after the second or third cycle. This finding may reflect the properties of the polyclonal antibody preparation used for these studies. Using such a polyclonal antibody column, very high affinity species of antibody may be blocked upon initial interaction with the antigen, whereas, during the first cycle, medium- and low-affinity species would both adsorb and release the antigen. Due to the harsh elution conditions used, the low affinity antibodies may eventually lose their binding capacity. Thus, after several cycles, the majority of available antibody species on the column could be expected to display intermediate affinity toward the antibody. After extended usage, the latter would also lose their binding capacity, and the column would eventually be unusable. It is also interesting to note that in this study columns containing egg white avidin were employed. Despite the valid claims (Bayer and Wilchek, 1980; Wilchek and Bayer, 1988) that the inherent alkalinity and presence of oligosaccharide residues are two potential sources of nonspecific binding, the observed levels of contaminating material in the purified antigen preparation (Fig. 2) were remarkably low. In some cases, however, it may be necessary to use columns containing bacterial streptavidin or modified avidin to obtain a product of increased purity. In the future, we are planning to use such columns for the ‘cascade isolation’ of proteins. For example, the avidin/biotinyl anti-transferrin/transferrin column described above can, in theory, be applied directly for the isolation of the transferrin receptor, the product of which can even be used to selectively fish out other proteins. Thus, the receptor-bound column may be used in the future to isolate monoclonal antibodies against the receptor or for other binding proteins which bind to the receptor in the cytosol (e.g., those which participate in post-endocytotic interactions). Acknowledgements The authors gratefully acknowledge the support of the Yeda Foundation, Rehovot, Israel.
REFERENCES Bayer, E. A., and Wilchek, M . (1978). Emerging techniques: the avidin/biotin complex as a tool in molecular biology. Trends Biochem. Sci. 3, N257-N259. Bayer, E. A., and Wilchek. M . (1980). The use of the avidin/biotin complex as a tool in molecular biology. Meth. Biochem. Anal. 26, 1 4 5 . Bayer, E. A., Ben-Hur, H., and Wilchek, M. (1987). Enzyme-based detection of glycoproteins on blot transfers using avidin/biotin technology. Anal. Biochem. 161,123-1 31. Buckie, J. W., and Cook, G. M. W. (1986). Specific isolation of surface glycoproteins from intact cells by biotinylated concanavalin A and immobilized streptavidin. Anal. Biochem. 156, 463472. 106 JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 3,1990
Cuatrecasas, P., Wilchek, M.. and Anfinsen, C. B. (1 968). Selective enzyme purification by affinity chromatography. R o c . Natl Acad. Sci. USA 61,636-643. Green, N. M . (1 970). Spectrophotometric determination of avidin and biotin. Methods Enzymol. 18A. 41 8 4 2 4 . Kohn, J., and Wilchek. M. (1982). Mechanism of activation of Sepharose and Sephadex by cyanogen bromide. Enzyme Microb. Technol. 4. 161-1 63. Kohn, J., and Wilchek, M . (1984). The use of cyanogen bromide and other novel cyanylating agents for the activation of polysaccharide resins. Appl. Biochem. Biotechnol. 9, 285-305. Mock, D. M., Lankford. G.. and Horowitz, P. (1 988). A study of the interaction of avidin with ANS as a probe of the biotin binding
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A V l D l N C O L U M N F O R I M M O B I L I Z A T I O N OF L I G A N D S site. Biochim. Biophys. Acta 956, 23-29. O’Shannessy, D. J.. Dobersen, M. J., and Charles, R. H. (1984). A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties. lmmunol. Lett. 8, 273-277. Updyke. T V., and Nicolson, G. L. (1984). lmmunoaffinity isolation of membrane antigens with biotinylated monoclonal antibodies and immobilized streptavidin matrices. J. Immunol. Methods 73,83-95. Wilchek, M., and Bayer, E. A. (1983). The avidin/biotin complex: a universal tool for biological systems. Qualityline 2. Wilchek, M., and Bayer, E. A. (1984). The avidin/biotin complex in immunology. lmmunol. Today5.3943. Wilchek, M., and Bayer, E. A. (1 988). The avidin/biotin complex in bioanalytical applications. Anal. Biochem. 171, 1-32. Wilchek, M., and Bayer. E. A. (1989). A universal affinity column using avidin/biotin technology. In Protein Recognition of Immobilized Ligands, ed. by T. W. Hutchens, pp. 83-90. Alan R. Liss. New York.
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Wilchek, M., and Gorecki., M. (1973). A new approach for the isolation of biologically active compounds by affinity chromatography: isolation of trypsin. FEBS Lett. 31, 149-1 52. Wilchek. M., and Miron, T. (1982). Immobilization of enzymes and affinity ligands onto agarose via stable and uncharged carbamate linkages. Biochem. lnternat. 4, 629-635. Wilchek, M., and Miron, T. (1985). Activation of Sepharose with N,N’-disuccinimidyl carbonate. Appl. Biochem. Biotech. 11, 191-1 93. Wilchek, M., Miron, T., and Kohn, J. (1984). Affinity chromatography. Methods Enzymol. 104.3-55. Wilchek, M., Oka, T., and Topper, Y. J. (1975). Structure of a soluble superactive insulin is revealed by the nature of the complex between cyanogen bromide-activated Sepharose and amines. Proc. Natl Acad. Sci. USA 72, 10551058. Received 26 January 1990; accepted (revised) 9 April 1990
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The avidin/biotin system was applied as a general mediator in the adsorption/desorption or immobilization of biologically active macromolecules to solid supports. In this context, model biotinylated proteins (lectins and antibodies) were attached to avidin-coupled Sepharose. As examples for affinity chromatography, peanut agglutinin and anti-bansferrin antibody were used to isolate asialofetuin and transferrin, respectively. The capacity and product yields were significantly better than those achieved with conventional affinity chromatography on CNBractivated Sepharose columns containing the same lectin or antibody. Moreover, the columns were characterized by improved stability properties exhibiting remarkably low levels of leakage.
INTRODUCTION Over the past two decades, the general trend for the purification of biologically active compounds has clearly favored the use of affinity chromatography (Wilchek et al., 1984). Using this technology, one usually binds a ligand chemically to a carrier matrix; the immobilized ligand then recognizes a biological target molecule to be purified. This approach is in many ways very appealing, but in laboratories where more than one product is routinely purified, many different types of affinity columns must be prepared and maintained. One of the difficult steps in preparing an affinity column is the chemical activation of the inert matrix prior to attachment of the biologically active ligand. Historically, cyanogen bromide activation of Sepharose was first employed for this purpose (Cuatrecasas et al., 1968). The original procedure is still widely in use today, despite the hazards involved in CNBr activation and innovative development of alternative activating reagents (Wilchek and Miron; 1982, 1985; Kohn and Wilchek, 1984). Moreover, the resultant isourea bond is unstable, thus causing leakage of the affinity ligand and consequent reduction in capacity and specificity (Wilchek et al., 1975; Kohn and Wilchek, 1982). In addition, storage of activated matrices consistently results in reduced levels of coupling compared to that of the freshly prepared affinity column. Many biochemists are uncomfortable with chemical reactions. It would therefore be convenient to have an activated carrier to which the ligand can be coupled Abbreviations used: Ab, anti-human transferrin antibody; B, biotinyl moiety: B-cap.but, biotinyl Nc-aminocaproyl-W-aminobutyryl moiety; BNHS, biotinyl N-hydroxysuccinimide ester; BHZ, biotin hydrazide; PBS, 10 m M phosphate buffered saline (pH 7.4); PNA, peanut agglutinin; SDS-PAGE, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. * Author to whom correspondence should be addressed.
biologically. An avidin-containing column is appropriate for these purposes (Bayer and Wilchek, 1978; Wilchek and Bayer, 1989). Alternatively, an antihapten antibody column with an association constant greater than lo8 M would also be suitable (Wilchek and Gorecki, 1973). In this context we have previously promoted the use of avidin/biotin technology for improving the versatility of affinity chromatography as a method in general (Bayer and Wilchek, 1980; Wilchek and Bayer, 1983; 1984). Such an ‘activated’ column would contain either egg white avidin or bacterial streptavidin to which biotinylated ligands can then be bound with exceptionally high stability. As we have suggested in the past (Wilchek and Bayer, 1988), virtually any ligand, including antibodies, binding proteins, enzymes, cofactors, inhibitors, drugs, hormones, nucleic acids, etc, can be easily biotinylated and bound to an avidin column. Many biotinylated biomolecules are commercially available. Such immobilized ligands would then be used for the purification of an appropriate molecular counterpart. A distinct advantage of this system would be that following preparation of the initial avidin-containing affinity matrix, additional chemical manipulations would not be required. Indeed, such an approach was used for the analytical isolation of cell surface antigens (Updyke and Nicolson, 1984) and lectins (Buckie and Cook, 1986). However, the system can also be exploited for the immobilization of precious biotinylated ligands which are available in very limited quantities as well as for the large scale preparation of desired biomolecules (e.g., antigens using monoclonal antibodies). In this study, we describe conditions for efficient immobilization of biotinylated lectins and antibodies to an avidin/Sepharose column. The resultant affinity columns were used to demonstrate a model for large scale purification of glycoproteins and antigens. Interestingly, the performance of these columns was far superior to that of the same affinity system prepared by conventional methodology, as reflected by the respective capacities, yields and lack of leakage.
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and containing about 1.6 mg transferrin per mL Sepharose) was washed sequentially with PBS, 0.2 M acetic acid and again with PBS. Rabbit anti-human transferrin antiserum (7 mL dissolved in an equivalent volume of PBS) was applied to the column, and the column was washed with PBS until the effluent fractions registered optical densities (280 nm) of less than 0.03. The eluant solution (0.2 M acetic acid) was then applied and the column was washed until the optical density dropped below 0.05. The fractions (65 mL total) were pooled, dialysed against PBS, centrifuged to remove precipitates, and concentrated in an Amicon ultrafiltration unit (Lexington, USA). The preparation was stored in 0.5 mL aliquots at a concentration of 2.3 mg/mL, total yield 13.7 mg.
EXPERIMENTAL Materials. The following materials were obtained from Sigma, St. Louis, MO, USA: biotin, iminobiotin, asialofetuin (type I ) fetuin (type IV), Coomassie brilliant blue R, and a high molecular weight standard kit containing the following markers: myosin (205 000), P-galactosidase (1 16 000), phosphorylase b (97 400), bovine serum albumin (BSA) (67 000), ovalbumin (45 000), and carbonic anhydrase (29 000). Egg white avidin was generously provided by S. C. Belovo (Bastogne, Belgium). Peanut agglutinin (PNA) was a generous gift by Professor Nathan Sharon of our department. Rabbit anti-human transferrin antiserum was a generous gift by Dr Harry Langbeheim of BioMakor, Inc. (Rehovot, Israel). Human plasma was obtained from the Magen David Adom Blood Bank (Jaffa, Israel). Preparation of avidin/Sepharose column. In the experiments described in this work avidin/Sepharose was prepared by the conventional CNBr-activation procedure. In later experiments, equivalent (and sometimes improved) results were obtained using either the 'cyano-transfer' activation procedure (Kohn and Wilchek, 1984) or chloroformate-activation of Sepharose (Wilchek and Miron, 1982). For stability considerations, the latter procedure is the preferred method for column preparation. The avidin columns can be stored at 4 "C for at least 5 years in the presence of 0.1 Yo sodium azide and can be used immediately after a short wash protocol using successive passages of equilibration and elution buffers. Preparation of biotinylated peanut agglutinin (B-PNA). In this work, PNA was used as a model binding protein. In initial studies, we were interested in calibrating the correlation between the number of biotin molecules attached to a representative binding protein and its subsequent attachment to an avidin column. For this purpose, varying molar ratios of biotinyl N hydroxysuccinimide ester (BNHS) were used to biotinylated peanut agglutinin (B-PNA). PNA, at a concentration of about 2.5 mg/mL was dissolved in a 0.1 M NaHC03 solution containing 0.2 M NaCI. The solution was centrifuged at 1500 x g for 10 min at 25 "C to remove precipitates. The supernatant fluids were collected and the optical density (280 nm) was measured. The solution was adjusted to 20 p~ (EL: = 7.7) protein. A 20 mM dimethyl formamide solution containing BNHS (6.8 mg/mL) was prepared, and 0,20,40,80 and 200 pL, respectively, were added to 4 mL samples of the prepared PNA solution (80 nmol PNA per sample). The final dimethyl formamide concentration of each sample was adjusted to 5%. Each reaction mixture was allowed to stand for 2 h at 25 "C, and each sample was dialysed separately, first against 0.15 M NaCl and then against PBS. The samples were collected and in each case about 90% of the initial protein could be recovered. The preparations were stored at -20 "C in 1 mL aliquots. Affinity isolation of anti-transferrin antibodies. A column (20 mL resin) containing Sepharose/transferrin (prepared by the conventional CNBr-activating procedure
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Preparation of biotinylated anti-transferrin antibodies. I , B-Ab. A solution of anti-transferrin antibody (3.4 mg in 1.5 mL) was treated with 50 pL BNHS solution (4 mg/mL in dimethyl formamide). The reaction was allowed to proceed for 2 h at 25 "C, and the reaction mixture was dialysed exhaustively against PBS. 2, Bcap.but-Ab. A dimethyl formamide solution (30 pL) of B-cap.but-NHS (10 mg/mL) was added to antitransferrin antibody (3.4 mg in 1.5 mL). After 2 h at 25 "C, the reaction mixture was dialysed exhaustively against PBS. 3, BHZ-Ab. Biotinylation of antitransferrin antibody was carried out by a slight modification of the procedure described by O'Shannessy et al. (1984). Antibody solution (3.4 mg in 1.5 mL PBS) was oxidized with 10 mM (final concentration) sodium periodate. The solution was dialysed for 4 h against PBS and 4 mg (solid) BHZ was added. After a 2 h incubation period at 25 "C, the reaction mixture was dialysed exhaustively against PBS. Preparation of affinity columns. Samples of the biotinylated
binding protein (lectin or antibody) were applied to the avidin column. The absorbance (Azxo)of the applied and effluent fractions were examined in order to estimate the extent of binding. We usually apply between 0.5- to 2-fold molar ratios of the biotinylated protein to avidin. One approach to establish the preferred ratio of the column constituents is to determine the number of free biotin binding sites before and after application of the biotinylated protein to the column. This can be accomplished by several methods, e.g., spectrophotometrically (Green, 1970), fluorometrically (Mock et ul., 1988) or using radioactive biotin obtained from either Amersham (Amersham, UK) or New England Nuclear (Dreiech, FRG). A second approach is to apply successive amounts of the biotinylated protein and to determine applied vs effluent readings of each application. In this case, initial applications of the biotinylated protein are usually adsorbed entirely by the avidin column; subsequent applications are only partially adsorbed. This approach gives an empirical indication of the capacity of the avidin column for a given biotinylated protein. It is usually advisable not to overload the column; a reasonable measure for an effective affinity column is to leave about two thirds of the original number of biotin binding sites or to load about one third of the column capacity with the desired biotinylated protein.
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Quantitative affinity chromatography. A small (0.5-1 .O mL) affinity column containing the desired resin was used in each case. 1 mL fractions were collected manually using a 1 mL volumetric flask. The amount of protein per fraction was usually determined spectrophotometrically, and the purity of the fractions was, in some cases, confirmed either by SDS-PAGE or by an appropriate dot-blot assay. In the case of the PNA columns, the adsorbed asialofetuin was released using a PBS solution containing 0.5 M galactose. In the case of the antibody column, a solution of 0.1 M acetic acid was used to release the antigen.
Gel electrophoresis. Polyacrylamide gel electrophoresis was
performed using 10% separating gels in the presence of sodium dodecyl sulfate (SDS-PAGE) under conditions described previously (Bayer et al., 1987).
RESULTS Biotinylated lectins In order to determine the feasibility of such systems, we first examined the immobilization characteristics of a simple purified binding protein for isolating a purified macromolecule. As a model system for this study, peanut agglutinin (PNA) was biotinylated, the biotinylated lectin (B-PNA) was bound to avidin-coupled Sepharose, and the product was used for the isolation of a commercial preparation of a galactose-containing glycoprotein (asialofetuin). PNA was subjected to biotinylation by conventional procedures using various molar ratios (relative to the concentration of protein) of biotinyl-N-hydroxysuccinimide ester (BNHS). Optimal binding to Sepharose/avidin affinity columns was achieved at an initial BNHS:PNA ratio of about 10: 1 (Fig. 1). The exact ratio of biotin moieties (average) per protein molecule was not determined in these experiments. In our experience, such an assessment is mainly of academic value. It is more relevant to gauge the interaction of a biotinylated preparation with an avidin probe. Again, experience has taught us that the interaction with an immobilized form of avidin is usually preferred, since the value obtained gives an indication of the percent of molecules in the preparation which will interact with the avidin-containing probe. On columns containing 1 mg avidin per mL Sepharose, 3.5 mg B-PNA could be readily attached. The capacity and elution efficiency of such columns for asialofetuin was much greater than those of conventional PNA-containing columns which were prepared using the standard CNBr-activation method. Samples of asialofetuin were loaded onto the two types of affinity column. In the case of the conventional PNA/Sepharose column ( I .7 mg protein per mL resin), the capacity was quite low; only 0.37 mg asialofetuin could be loaded onto 1 mL of the column. A more 104 JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 3 , 1 9 9 0
100
=0
75
c
c
a, t
50 8
25
r :7 A
Figure 1. Interaction of biotinylated PNA preparations with avidin/ Sepharose column. Purified samples of PNA were biotinylated using various molar ratios of BNHS. Following dialysis, the respective biotinylated preparations were applied to an avidin column and the percent retained (percent protein applied minus percent in effluent) was determined.
serious problem, however, was the finding that only 0.24 mg of the glycoprotein could be selectively released from the column using 0.5 M galactose, indicating that about a third of the target material was very tightly bound. Thus, conventional PNA/Sepharose is a relatively ineffective affinity column. On the other hand, the performance of the PNA column prepared using avidin/biotin technology was excellent. The capacity of the column was comparatively high (1.2 mg per mL column) and the entire amount could be released selectively using the competing sugar. The eluted protein appeared pure by SDS-PAGE and by selective labeling of the blotted sample (data not shown). The product was not contaminated by detectable levels of either B-PNA or avidin. The results suggest that more effective affinity columns may be achieved through aviding/biotin mediation. Attempts were made to release the B-PNA from the Sepharose/avidin column, such that the latter would be reusable. The method comprised elution of the biotinylated lectin using solutions of iminobiotin at pH 1 1. About 70% of the biotinylated lectin could be recovered in this manner.
Biotinylated antibodies The results described above for biotinylated lectins were extended for application to biotinylated antibodies. Commercially prepared (BioMakor, Rehovot, Israel) polyclonal antibodies, elicited against human transferrin, were used as a model system. The characteristics of three different biotinylating reagents were compared for effectiveness in incorporating biotin into the antibody molecule. These were: 1, BNHS; 2, a longer chained derivative, biotinyl NC-aminocaproyl-Nuaminobutyryl-N-hydroxy-succinimideester B-cap.but NHS); and 3, biotin hydrazide (BHZ). The first two labels were used to biotinylate amino groups of the antibodies and the hydrazide derivative was used to label the sugar residues of the periodate oxidized antibody.
0 1990 by John Wiley & Sons, Ltd.
AVIDIN COLUMN FOR IMMOBILIZATION OF LIGANDS
The capacity of the avidin column for the antibody preparations biotinylated via amino groups was greater than that observed for the BHZ-derivatized antibody (Table 1 ). A typical column contained 1.5 mg avidin per mL Sepharose, and about 1.3 mg/mL B-Ab. All three types of the columns could be used to isolate transferrin (Table 2); the capacity of the columns was about 0.4 mg transferrin per mL Sepharose. A new column was prepared which contained 2 mg B-Ab per 1 mL Sepharose/avidin. Human plasma (diluted fourfold with PBS) was applied directly to this column. After extensive washing of this column, the adsorbed transferrin was released using 0.1 M acetic acid. Eluant fractions were collected, and the column was regenerated by washing with PBS. A second sample of serum was applied and eluted in an identical fashion. Absorbance measurements of the eluted fractions indicated that about 0.9 mg was purified initially and another 0.75 mg sample was purified upon regeneration of the column. Subsequent trials showed a stabilization of the column, and about 0.7 mg per mL column was consistently isolated per cycle. Both preparations were equally pure by SDS-PAGE (Fig. 2). Only very low levels of other contaminating serum proteins could be detected. The purified transferrin appeared to contain even less contaminating antibody than that of the commercially available (Sigma) protein (against which the
Table 1 . Binding of biotinylated anti-transferrin preparations to avidin/Sepharose B~Ab
Applied Effluent Bound initially AcOH wash Total bound
0.85 0.02 0.83 0.15 0.68
6-cap.but-Ab
0.67 0.01 0.66 0.06 0.60
BHZ~Ab
0.75 0.31 0.44 0.21 0.23
Values represent amount (mg) of the designated biotinylated antibody preparation which was present in each fraction. Column volumes of 0 5 mL were used in each case.
Table 2. Isolation of commercial preparation of transferrin on biotinylated antibody bound to avidin/Sepharose 6-Ab
6-cap but-Ab
BHZ Ab
0.50 0.16 0 34 0.20
0.50 0.27 0.23 0.18
0.50 0.36 0.14 0.15
0.45 0.13 0.28 0.17
0.47 0.22 0.25 0.22
NT
Trial 1
Applied Effluent Bound AcOH eluant Trial 2
Applied Eff Iuent Bound AcOH eluant
~
-
Values represent the amount (mg) of transferrin contained in each fraction. Column volumes of 0.5 mL were used in each case. NT, not tested.
$3 1990 by John
Wiley & Sons, Ltd.
A
B
C
D
Figure 2. SDS-PAGE of transferrin purified directly from human plasma on a B-Ab column. Lane A shows the pattern of human plasma, a sample of which was applied to the column; lane B shows the pattern of transferrin purified on the B-Ab/avidin/Sepharose affinity column; lane C gives the pattern of Sigma transferrin; and lane D shows the position of molecular weight protein standards (205 000, 116 000, 97 400, 67 000, 45 000 and 29 OOO), Samples of 30 pL were added to each well.
anti-transferrin antibodies used in this study were prepared).
DISCUSSION We have shown that avidin columns can be used to bind different biotinylated proteins and that the resultant affinity column is appropriate for the selective isolation of biologically active target molecules from crude material. The biotinylation step is very easy to perform, and most biotinylated proteins can be stored in the active state indefinitely. In this sense, the avidin column is a ‘preactivated’ column, and the biotinylated protein can be attached within minutes. In applying this approach, we have demonstrated the use of immobilized proteins for the isolation of their biological counterparts, e.g., biotinylated antitransferrin for transferrin and biotinylated PNA for asialofetuin. In each case, the target protein could be eluted from the column, and the affinity column could be reused. The isolated protein was remarkably pure as tested by SDS-PAGE. The columns proved to be more efficient than standard affinity chromatographic techniques (on conventional columns). Using a polyclonal antibody preparation against human transferrin, we examined three different biotinylation procedures to determine their effect on JOURNAL OF MOLECULAR
RECOGNITION, VOL. 3, No. 3,1990 105
EDWARD A. BAYER A N D MEIR WILCHEK
purification of the model antigen. I t was clear that higher levels of biotinylation could be obtained by biotinylating via the amino groups o f the antibody compared to that achieved through periodate oxidation of the sugars. Using the latter procedure (coupling of biotin hydrazide to periodate-generated sugar aldehydes o f the antibody), about half of the amount of biotinylated antibody which was initially bound to the column was released upon washing with acetic acid. Immobilization achieved through biotinylation via the peptide linkage was more stable (see Table 2). The best results were achieved using a long chain reagent bound to the amino groups of the antibody. In the case of B-cap.but-Ab, the stability of the resultant avidinjsepharose affinity column to the acetic acid washing step was greater than that of the shorter chained B-Ah. In this work, however, we did not attempt to couple a long chain biotin-containing derivative (e.g., biocytin hydrazide) through the sugar residue of the antibody; consequently, we cannot state conclusively whether the reduced levels of binding observed with the biocytin hydrazide conjugate is solely related to the chemistry of biotinylation via the sugar or whether a long chain derivative of biocytin hydrazide would have improved the stability characteristics or capacity of the column. In any event, it should be noted that all three of the resultant immunoaffinity columns could be used successfully to isolate the antigen in question. Once the biotinylated binding protein is applied to the avidin column and subjected to the stringent washing procedure, the affinity column is remarkably stable. In this regard, it is advised to apply subsaturating amounts of the biotinylated protein such that a population of residual free biotin binding sites will remain. These would then be available to crosslink biotinylated protein which may disengage from the avidin matrix due to the low but finite dissociation constant. More recent work in our laboratory has shown that avidin coupled to Sepharose using the chloroformate activation procedure is preferred to that coupled via CNBr. Nevertheless, even using CNBr-induced coupling of avidin, the resultant columns were remarkably stable compared to conventional antibody columns prepared by direct CNBr linkage. The addition of the biotinylated protein thus seems to improve the leakage characteristics
of the column, perhaps due to crosslinking among the avidin molecules. We noted also that the capacity of the columns was somewhat reduced after the first trial, reaching a relatively stable plateau after the second or third cycle. This finding may reflect the properties of the polyclonal antibody preparation used for these studies. Using such a polyclonal antibody column, very high affinity species of antibody may be blocked upon initial interaction with the antigen, whereas, during the first cycle, medium- and low-affinity species would both adsorb and release the antigen. Due to the harsh elution conditions used, the low affinity antibodies may eventually lose their binding capacity. Thus, after several cycles, the majority of available antibody species on the column could be expected to display intermediate affinity toward the antibody. After extended usage, the latter would also lose their binding capacity, and the column would eventually be unusable. It is also interesting to note that in this study columns containing egg white avidin were employed. Despite the valid claims (Bayer and Wilchek, 1980; Wilchek and Bayer, 1988) that the inherent alkalinity and presence of oligosaccharide residues are two potential sources of nonspecific binding, the observed levels of contaminating material in the purified antigen preparation (Fig. 2) were remarkably low. In some cases, however, it may be necessary to use columns containing bacterial streptavidin or modified avidin to obtain a product of increased purity. In the future, we are planning to use such columns for the ‘cascade isolation’ of proteins. For example, the avidin/biotinyl anti-transferrin/transferrin column described above can, in theory, be applied directly for the isolation of the transferrin receptor, the product of which can even be used to selectively fish out other proteins. Thus, the receptor-bound column may be used in the future to isolate monoclonal antibodies against the receptor or for other binding proteins which bind to the receptor in the cytosol (e.g., those which participate in post-endocytotic interactions). Acknowledgements The authors gratefully acknowledge the support of the Yeda Foundation, Rehovot, Israel.
REFERENCES Bayer, E. A., and Wilchek, M . (1978). Emerging techniques: the avidin/biotin complex as a tool in molecular biology. Trends Biochem. Sci. 3, N257-N259. Bayer, E. A., and Wilchek. M . (1980). The use of the avidin/biotin complex as a tool in molecular biology. Meth. Biochem. Anal. 26, 1 4 5 . Bayer, E. A., Ben-Hur, H., and Wilchek, M. (1987). Enzyme-based detection of glycoproteins on blot transfers using avidin/biotin technology. Anal. Biochem. 161,123-1 31. Buckie, J. W., and Cook, G. M. W. (1986). Specific isolation of surface glycoproteins from intact cells by biotinylated concanavalin A and immobilized streptavidin. Anal. Biochem. 156, 463472. 106 JOURNAL OF MOLECULAR RECOGNITION, VOL. 3, No. 3,1990
Cuatrecasas, P., Wilchek, M.. and Anfinsen, C. B. (1 968). Selective enzyme purification by affinity chromatography. R o c . Natl Acad. Sci. USA 61,636-643. Green, N. M . (1 970). Spectrophotometric determination of avidin and biotin. Methods Enzymol. 18A. 41 8 4 2 4 . Kohn, J., and Wilchek. M. (1982). Mechanism of activation of Sepharose and Sephadex by cyanogen bromide. Enzyme Microb. Technol. 4. 161-1 63. Kohn, J., and Wilchek, M . (1984). The use of cyanogen bromide and other novel cyanylating agents for the activation of polysaccharide resins. Appl. Biochem. Biotechnol. 9, 285-305. Mock, D. M., Lankford. G.. and Horowitz, P. (1 988). A study of the interaction of avidin with ANS as a probe of the biotin binding
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A V l D l N C O L U M N F O R I M M O B I L I Z A T I O N OF L I G A N D S site. Biochim. Biophys. Acta 956, 23-29. O’Shannessy, D. J.. Dobersen, M. J., and Charles, R. H. (1984). A novel procedure for labeling immunoglobulins by conjugation to oligosaccharide moieties. lmmunol. Lett. 8, 273-277. Updyke. T V., and Nicolson, G. L. (1984). lmmunoaffinity isolation of membrane antigens with biotinylated monoclonal antibodies and immobilized streptavidin matrices. J. Immunol. Methods 73,83-95. Wilchek, M., and Bayer, E. A. (1983). The avidin/biotin complex: a universal tool for biological systems. Qualityline 2. Wilchek, M., and Bayer, E. A. (1984). The avidin/biotin complex in immunology. lmmunol. Today5.3943. Wilchek, M., and Bayer, E. A. (1 988). The avidin/biotin complex in bioanalytical applications. Anal. Biochem. 171, 1-32. Wilchek, M., and Bayer. E. A. (1989). A universal affinity column using avidin/biotin technology. In Protein Recognition of Immobilized Ligands, ed. by T. W. Hutchens, pp. 83-90. Alan R. Liss. New York.
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Wilchek, M., and Gorecki., M. (1973). A new approach for the isolation of biologically active compounds by affinity chromatography: isolation of trypsin. FEBS Lett. 31, 149-1 52. Wilchek. M., and Miron, T. (1982). Immobilization of enzymes and affinity ligands onto agarose via stable and uncharged carbamate linkages. Biochem. lnternat. 4, 629-635. Wilchek, M., and Miron, T. (1985). Activation of Sepharose with N,N’-disuccinimidyl carbonate. Appl. Biochem. Biotech. 11, 191-1 93. Wilchek, M., Miron, T., and Kohn, J. (1984). Affinity chromatography. Methods Enzymol. 104.3-55. Wilchek, M., Oka, T., and Topper, Y. J. (1975). Structure of a soluble superactive insulin is revealed by the nature of the complex between cyanogen bromide-activated Sepharose and amines. Proc. Natl Acad. Sci. USA 72, 10551058. Received 26 January 1990; accepted (revised) 9 April 1990
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