Solid Phase Assembly of Defined Protein Conjugates - American

958

Bioconjugate Chem. 2002, 13, 958−965

Solid Phase Assembly of Defined Protein Conjugates John C. Russell,* Tracey L. Colpitts, Shelley R. Holets-McCormack, Thomas G. Spring, Stephen D. Stroupe, Andrew D. Vogt, and Sie-Ting Wong D09FQ,

AP20,

Abbott

Laboratories,

100

Abbott

Park

Road,

Abbott

Park,

Illinois

60064-6015.

Received April 26, 2002; Revised Manuscript Received June 25, 2002

We have developed a solid-phase procedure for protein-protein conjugation that gives greater control over product size and composition than previous methods. Conjugates are assembled by sequential addition of activated proteins to the support under conditions suitable for maintaining the activity of the proteins. The total number of conjugate units to be prepared is fixed in the first step by the quantity of the first protein absorbed by the support. In each following step, the added protein links only to previously bound protein. The final conjugate is released to solution by cleaving the linker holding the first protein to the support. This stepwise assembly provides uniformly sized conjugates of the desired size and composition with placement of components at the desired positions within the structure. Using this approach, we have prepared a series of conjugates containing R-phycoerythrin as the central protein, with varying quantities of alkaline phosphatase and IgG with expected molecular masses ranging from 1.6 to 11.5 MDa. Size-exclusion chromatography and atomic force microscopy demonstrate homogeneity and control of the conjugate size. In an immunoassay for human thyroid stimulating hormone, the conjugates show signals consistent with their compositions.

INTRODUCTION

There are numerous occasions in life sciences in which it is desirable to link two or more proteins together to obtain a species that combines the properties of each. Most commonly, a protein with specific binding affinity to a target is linked to a second protein with a desired activity. In immunodiagnostic applications, the binding protein is typically an antibody directed against the desired analyte, and the second protein is usually a fluorescent protein or an enzyme that generates a signal detectable by eye or by instrumentation (1-4). In therapeutic applications, the binding protein may be an antibody or a ligand with specific binding to a cell surface receptor, and the second protein may be a toxin that kills the targeted cell (5). A variety of methods have been devised to link proteins together. Most commonly, the two proteins are chemically activated so that groups on one can form stable covalent links to groups on the other. They are then combined in a homogeneous solution and allowed to react under conditions suitable for both linking and maintenance of their activities. When a sufficient proportion of the proteins are linked, the reaction is stopped by adding reagents that deactivate one or both of the reactive groups. These conjugation reactions are difficult to control. The activating agents usually generate numerous active positions on each protein molecule, with the result that many points of attachment are possible. Linkage at one active group on a protein does not prevent linkage at a second or third point, and a broad distribution of aggregates consisting of many units of each of the proteins may result. While it is frequently desirable to produce multimeric conjugates, it is also desirable to control these so that a majority of the species produced contains the number of each protein unit most suitable for the intended purpose of the conjugate. We have developed a method for preparing protein-protein con* Address correspondence to this author. E-mail: john.c.russell@ abbott.com.

jugates with highly predictable and controllable composition and size. A first protein, termed the “core”, is bound to a solid support via a cleavable linker. Additional proteins are introduced sequentially as “layers” under conditions that allow their stable linkage only to the core or to the immediately previous layer of protein. After completion of the reaction sequence, the protein assembly is released by cleaving the linker holding the core protein to the support. Immobilization of the core protein on the support prevents interaction of the assembly with other immobilized assemblies during the reaction sequence. Therefore, at each step, the assembly can grow only by linking the added free protein. If the added proteins are not self-reactive, the growth at each step is limited to the quantity that can directly link to the previous layer. Excess can be added to saturate all available sites and unreacted material washed from the support before the next step is started. Each successive layer contains more protein than the previous layer, suggesting radial growth of the conjugates. Very large conjugates can be prepared with relatively few steps of protein addition. This method of protein conjugation has several advantages over solution methods. The narrow distribution of sizes of the released conjugate allows it to be optimized with respect to intended properties. The sequence of addition allows placement of specific components at the most desirable position in the conjugate. Binding agents may be added in the final conjugation step, placing them at the periphery of the conjugate where they can most effectively interact with their target. Other proteins beyond the binding and reporter proteins may be included to confer additional properties. The ability to control the quantities and positions of proteins within the assemblies leads us to term them “defined conjugates”. In the present work, we use the solid-phase technique to prepare a number of defined conjugates containing

10.1021/bc0255438 CCC: $22.00 © 2002 American Chemical Society Published on Web 08/14/2002

Solid Phase Assembly of Defined Protein Conjugates

R-phycoerythrin (RPE)1 as the core, several layers of alkaline phosphatase (AP), and a final layer consisting of antibody specific to the R subunit of thyroid stimulating hormone (Ab). We then compare these with conventionally prepared conjugates using HPLC and atomic force microscopy to compare size and dispersity. The defined conjugates are then tested in an immunoassay format to demonstrate the dependency of enzyme and antibody content on their ability to generate signal. Scheme 1 diagrams the process of controlled conjugation used in this report. An agarose support is oxidized with periodate to give immobilized aldehydes (6). The hydrazide function of the heterobifunctional linker N-(maleimidocaproic acid)hydrazide (EMCH) reacts with the aldehydes to give a maleimide group linked to the support via the hydrazone. Proteins to be conjugated are activated with N-succinimidyl S-acetylthioacetate (SATA) to give sulfhydryl groups or with γ-maleimidobutyric acid Nhydroxysuccinimide ester (GMBS) to give maleimide groups. A sulfhydryl-activated protein reacts with the maleimide of the bound EMCH, forming a stable thioether linkage, still held to the agarose support. Remaining maleimide groups on the support are destroyed by adding mercaptoethanesulfonic acid sodium salt (MESNA). The support is then washed to remove excess MESNA, leaving sulfhydryl groups only on the bound protein. After washing the agarose to remove unbound reactive materials, a second protein, containing maleimide groups, is added. This reacts with remaining sulfhydryl groups on the first protein, again forming a thioether linkage. If an excess of the second protein is added, all the available binding locations on the first protein will be occupied. Remaining sulfhydryl groups on the first protein will be available for reaction only with small molecules. The exposed surface of the growing conjugate consists of the second protein with maleimide groups available for reaction with a third protein (or a repeat of the first) containing sulfhydryl groups. When all the desired layers of protein have been added, the remaining thiol groups are converted to unreactive thioethers with N-ethylmaleimide and the remaining maleimide groups likewise capped with MESNA. The conjugate is then released from the support by adding hydroxylamine. This reacts with the linkage holding the conjugate to the support and releases it, presumably as the hydrazide. EXPERIMENTAL SECTION

Materials. R-Phycoerythrin was obtained from Prozyme (San Leandro, CA). Calf intestinal alkaline phosphatase was obtained from Boehringer Mannheim (Indianapolis, IN). AntiTSHalpha IgG (MIT 0414) was obtained from Genzyme (Framingham, MA). Sodium periodate, Sepharose CL2B, N-ethylmaleimide, mercaptoethanesulfonic acid sodium salt, 5,5′-dithiobis(2-nitrobenzoic acid), tris(hydroxymethyl)aminomethane, 3-[(3-cholamidopropyl)1 Abbreviations: RPE, R-phycoerythrin; AP, calf intestinal alkaline phosphatase; Ab, antibody; EMCH, N-(-maleimidocaproic acid)hydrazide; MESNA, mercaptoethanesulfonic acid sodium salt; GMBS, γ-maleimidobutyric acid N-hydroxysuccinimide ester; SATA, N-succinimidyl S-acetylthioacetate; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); Tris, tris(hydroxymethyl)aminomethane; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate; PBS, 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2; EDTA, ethylenediaminetetraacetic acid; PCE, PBS containing 2 mg/mL CHAPS and 5 mM EDTA; TC75E, 100 mM Tris with 2 mg/mL CHAPS and 5 mM EDTA, pH 7.5; TC75E, 100 mM Tris with 2 mg/mL CHAPS and 5 mM EDTA, pH 7.5; TC8E, 100 mM Tris with 2 mg/mL CHAPS and 5 mM EDTA, pH 8.0.

Bioconjugate Chem., Vol. 13, No. 5, 2002 959

dimethylammonio]-1-propanesulfonate, triethanolamine hydrochloride, and γ-maleimidobutyric acid N-hydroxysuccinimide ester were obtained from Sigma (St. Louis, MO). N-Succinimidyl S-acetylthioacetate, N-(-maleimidocaproic acid)hydrazide, p-nitrophenyl phosphate substrate, Superblock blocking agent, and BCA protein assay kit were obtained from Pierce (Rockford, IL). Hydroxylamine 50% solution was obtained from Aldrich (Milwaukee, WI). The monoclonal antibody against the β subunit of human thyroid stimulating hormone (TSH) and standard solutions with known concentrations of TSH were obtained from Abbott Laboratories (North Chicago, IL). Activation of Proteins. (A) RPE-SH. To 200 µL of R-phycoerythrin at 10 mg/mL in 100 mM triethanolamine hydrochloride at pH 7.6 was added 10 µL of 100 mM N-succinimidyl S-acetylthioacetate (SATA) (7) in dimethylformamide. After 1 h incubation at room temperature, 10 µL of 50% hydroxylamine was added. After 40 min at room temperature, the mixture was desalted on Sephadex G25 into PBS with 5 mM EDTA, collecting 550 µL. The concentration was determined by the absorbance at 565 nm to be 10.1 µM (9), and the number of SH groups per molecule was determined to be 22.5 using Ellman’s reagent. (B) AP-SH. To 500 µL of alkaline phosphatase at 10 mg/mL in triethanolamine HCl was added 50 µL of 1 M sodium phosphate, pH 7.5, and 25 µL of 100 mM SATA in dimethylformamide. After 1 h incubation at room temperature, 25 µL of 50% hydroxylamine was added. After 40 min at room temperature, the mixture was desalted on Sephadex G25 into PBS with 5 mM EDTA, collecting 1.2 mL. The concentration was determined by the absorbance at 280 nm to be 30.5 µM, and the number of SH groups per molecule was determined to be 15.3 using Ellman’s reagent. According to the supplier, calf intestinal alkaline phosphatase contains 18 conjugatable amine groups. (C) AP-Mal. To 500 µL of alkaline phosphatase at 10 mg/mL in triethanolamine HCl was added 50 µL of 1 M sodium phosphate, pH 7.5, and 25 µL of 100 mM GMBS (8) in dimethylformamide. After 100 min at room temperature, the mixture was desalted on Sephadex G25 into PBS with 5 mM EDTA, collecting 1.2 mL. The concentration was determined by the absorbance at 280 nm to be 29.8 µM, and the number of maleimide groups per molecule was determined to be 15.4 by the change in absorbance at 300 nm after addition of MESNA to destroy the maleimide chromophore. (D) Ab-Mal. To 200 µL of Anti-TSHalpha IgG at 7.03 mg/mL in PBS was added 10 µL of 100 mM GMBS in dimethylformamide and 2 µL of 1 M sodium carbonate to give pH 7.7. After 100 min incubation at room temperature, the mixture was desalted into PBS with 5 mM EDTA, collecting 550 µL. The concentration was determined by the absorbance at 280 nm to be 15.9 µM, and the number of maleimide groups per molecule was determined to be 28.5 by the change in absorbance at 300 nm after addition of MESNA to destroy the maleimide chromophore. (E) Ab-SH. To 100 µL of Anti-TSHalpha IgG at 7.03 mg/mL in 100 mM sodium phosphate 150 mM sodium chloride, pH 7.2, was added 5 µL of 100 mM SATA in DMF and 1 µL of 1 M sodium carbonate. After 1 h incubation at room temperature, 10 µL of 50% hydroxylamine was added. After 40 min at room temperature, the mixture was desalted into PBS with 5 mM EDTA, collecting 550 µL. The concentration was determined by the absorbance at 280 nm to be 11.7 µM, and the number

960 Bioconjugate Chem., Vol. 13, No. 5, 2002

Russell et al.

Scheme 1. Reaction Sequence in Defined Conjugation

of SH groups per molecule was determined to be 25.4 using Ellman’s reagent. Preparation of Support. (A) Periodate Oxidation of Agarose. Approximately 20 mL of Sepharose CL2B was poured into a column fitted with a fritted disk, washed with water, and suspended in 20 mL of excess water. To this was added 200 µL of 100 mM sodium periodate and the mixture inverted several times to mix. After 75 min at room temperature, 1 mL of glycerol was

added and the mixture inverted several times to mix. After 15 min, the column was drained and washed with several column volumes of water and then with PBS. (B) Activation of Oxidized Agarose with EMCH. To a 20 mL column was added 6.0 mL of oxidized agarose. This was washed with 20 mL of PCE (see list of abbreviations). The washed support was vortexed with 3 mL of PCE and 90 µL of 100 mM EMCH in dimethylformamide. After 30 min incubation at room temperature,



Table 1. Quantities of Activated Proteins Added (italic) and Bound (bold) in Each Step of Conjugation step: reactant: added:

1 RPE-SH (nmol) 0.200

2 AP-Mal (nmol) 0.800

3 AP-SH (nmol) 1.80

reaction A B C D E F G H

bound 0.176 0.172 0.173 0.168 0.174 0.178 0.171 0.176

0.609 0.594 0.624 0.633 0.633 0.635 0.635 0.636

1.27 1.30 1.25 1.26 1.23 1.25 1.27


2.37 2.38 2.36 2.40 2.31 2.37

5 AP-SH (nmol) 4.50

3.62 3.65 3.66 3.57 3.63


Ab

Ab-SH Ab-Mal Ab-SH Ab-Mal Ab-Mal Ab-Mal Ab-Mal Ab-SH

5.15

Ab added

Ab bound

(nmol)

(nmol)

1.00 1.00 1.00 1.00 0.40 3.00 10.00 1.00

0.97 1.00 0.99 0.98 0.39 2.85 4.41 1.00

Table 2. Characteristics of Defined Conjugates concentration

A B C D E F G H

AP/RPE

Ab/RPE

molecular mass (kDa)

3.5 10.8 24.8 46.9 45.4 44.4 45.5 74.1

5.5 5.8 5.7 5.8 2.2 16.0 25.8 5.7

1551 2625 4566 7670 6932 8866 10476 11470

the column was drained, washed with 15 mL of cold TC75E (100 mM Tris, 0.2% CHAPS, 5 mM EDTA, pH 7.5), and placed in ice. Conjugation on Support. (A) Immobilization of RPE-SH. To each of eight 2 mL columns (reactions A-H of Table 1) was added 600 µL of the EMCH-treated resin, and the mixtures were washed with 2 mL of TC75E. The columns were cooled in ice; then 0.200 nmol (198 µL) of RPE-SH was added to each, and the resins were thoroughly dispersed by vortexing. After 20 min incubation in ice with occasional vortexing, 10 µL of 100 mM MESNA was added to each. After 5 min incubation in ice, the columns were drained and washed with 3.0 mL of cold TC8E. The absorbance of the first 1.5 mL of the effluent was measured at 565 nm to determine the quantity of RPE-SH in the effluent. This was subtracted from that originally added to determine the quantity bound to the support, shown in Table 1. (B) Addition of AP-Mal and AP-SH to Immobilized RPE-SH. Except for time spent draining and washing, the columns were kept in an ice bath throughout the conjugation procedure. The quantity of activated protein indicated in Table 1 was added to each, along with 100 µL of 1 M magnesium chloride and sufficient TC8E for complete dispersal of the resin on vortexing. The columns were incubated in ice 30 min with occasional vortexing and then drained, and the effluent was recycled through the column for a total of approximately 1 column volume. The columns were then washed with TC8E, and the next addition of activated protein was performed. The absorbance at 280 nm of the effluents was measured to determine the quantity of remaining alkaline phosphatase [(280) ) 140 000], shown in Table 1. (C) Addition of Ab-Mal and Ab-SH to Immobilized Conjugate. After the final addition and incubation of activated alkaline phosphatase according to Table 1, the columns were washed with 1.5 mL of TC75E and cooled in ice. The indicated quantity of activated Ab was added, the mixtures were incubated 5 min to allow dispersal of the protein, then 100 µL of 1 M magnesium chloride was added, and the mixture was incubated 30 min in ice. The columns were then drained and washed with 1.5 mL of

% recovery

by RPE (nM)

calculated (µg/mL)

BCA (µg/mL)

76.0 70.2 62.4 56.1 58.2 54.5 52.5 50.0

53.6 48.3 43.3 37.8 40.5 38.9 35.8 35.2

83.1 126.7 197.7 289.7 280.9 344.5 375.5 403.8

76.8 117.2 175.3 251.1 255.7 294.4 316.6 376.8

TC8E. The absorbance at 280 nm of the effluents was measured, and the quantity of Ab remaining in the effluent was calculated using (280) ) 210 000. From this, the quantity of Ab bound to the resin was calculated, and is shown in Table 1. (D) Release of Conjugates from Support. After washing off the unbound Ab, the resins were treated with 5 µL of 100 mM N-ethylmaleimide to deactivate residual SH groups. The columns were left in ice until protein addition and washing was complete for all of the preparations. To deactivate residual maleimide groups, 10 µL of 100 mM MESNA was added, the mixtures were vortexed, and the columns were returned to the ice bath. After 10 min, 20 µL of 50% hydroxylamine was added, and the mixtures were incubated 60 min at room temperature. The columns were then drained directly into desalting columns equilibrated with PBS and the products washed through with TC8E and PBS collecting 2.5 mL of product. Concentrations of product, shown in Table 2 , were calculated based on the absorbance at 565 nm, and yields were calculated based on recovery of RPE color. Protein concentrations of the conjugates were measured by the BCA protein assay using the enhanced protocol according to the manufacturers’ instructions. Alkaline phosphatase was used as the protein standard. Conjugation of Ab-SH and AP-Mal in Solution. To a solution of PBS with 5 mM EDTA was added 2 nmol of Ab-SH and 4 nmol of AP-Mal prepared as above to give a total 800 µL and the mixture incubated in an ice bath. Aliquots of 200 µL of the reaction mixture were mixed with 4 µL of 100 mM aqueous N-ethylmaleimide to stop the conjugations at times of 15, 60, 150, and 300 min. Characterization of Conjugates. (A) HPLC. To 300 µL of each conjugate was added 30 µL of 10 mg/mL CHAPS in PBS. A sample of 20 µL was passed through a Whatman Macrosphere GPC 1000A 250 × 4.6 mm column with 1 mg/mL CHAPS in PBS as the mobile phase at 0.2 mL/min, monitoring at 280 nm and 566 nm with a diode array detector.


(B) Atomic Force Microscopy. Silicon wafers (International Wafer) were cleaned by immersion in 3% hydrogen peroxide in sulfuric acid at 100 °C for 10 min, then washed with distilled water and blown dry. Samples were prepared by exposing 8 mm squares of the silicon wafers to dilutions of the conjugates at 1:100 in 10 mM sodium phosphate, 150 mM sodium chloride, pH 7.2. After 30 min at room temperature, the wafers were washed with water, blown dry with air, and heated at 45 °C for 2 h. The wafers were imaged using a NanoScope IIIa (Digital Instruments) atomic force microscope. Images were collected in TappingMode at ambient temperature in air using silicon-etched tips with a resonance frequency of 275-395 kHz and a scan rate of 1 µm/s. Tips were changed between samples to eliminate crosscontamination. (C) Comparison of Conjugates in TSH Assay. Wells of a 96-well microtiter plate were coated with a mAb directed against the β subunit of TSH at 20 µg/mL in PBS for 60 min at 37 °C and blocked with Superblock according to the manufacturers’ instructions. To the wells was added 25 µL of TSH standards, and the plate was covered and incubated 3 h at 37 °C. The wells were drained and washed 5× with water, and 100 µL of conjugate in 50 mM bis-tris-propane, 150 mM NaCl, 10 mM MgCl2, 1 mM ZnCl2, pH 7.2, containing 10% Superblock was added. The conjugate concentration was 40 pM RPE for one test and 200 ng/mL protein for another test. After 3 h at 37 °C, the wells were drained and washed 5× with water. One hundred microliters of substrate (PNPP, Pierce) was added, the plate placed at 37 °C, and the absorbance read at 405 nm at 30 s intervals over 30 min. Vmax (mA/min) was reported for each well. RESULTS

Yields of released conjugate, shown in Table 2, are based on recovery of bound RPE, measured by its absorbance at 565 nm. These range from 76% for conjugate A, with a single layer of AP, to 50% for conjugate H, with five layers. The remainder is visible as pink color remaining in the support. Very little additional conjugate was released from the supports even with prolonged exposure to hydroxylamine. Table 2 shows the molar ratios of AP to RPE and Ab to RPE absorbed by the support in the defined conjugate preparations. In reactions A, B, C, D, and H, the AP absorbed ranges from 3.5 to 74.1 per RPE, and the Ab is nearly constant at 5.5-5.8. For reactions D, E, F, and G, the AP is nearly constant at 44.4-46.9, and the Ab varies from 2.2 to 25.8. The average molecular mass for each of the assemblies was calculated from the uptake of the activated proteins, assuming a single RPE core in each discrete unit of conjugate and using molecular masses of 240 kDa for RPE, 140 kDa for AP, and 150 kDa for Ab. The molecular masses range from about 1.6 MDa for A to about 11 MDa for H. Molar concentrations of product are used with the molecular masses to calculate the mass concentrations. These compare well with mass concentrations measured by protein assay. HPLC traces of both defined and solution-phase conjugates on a size-exclusion column are shown in Figure 1. Peaks corresponding to the defined conjugates with more layers of AP elute earlier than those with fewer layers, consistent with the calculated molecular masses. Monitoring at 565 nm shows a decrease in peak intensity with larger conjugate size, reflecting the lower RPE concentrations, while monitoring at 280 nm shows an increase in peak intensity resulting from the higher levels

Russell et al.

Figure 1. HPLC of conjugates Top: defined conjugates A-H, and native RPE and AP, monitoring at 565 and 280 nm. Bottom: solution-phase conjugates K15, K60, K150, and K300, monitoring at 280 nm.

of AP on the larger conjugates. In contrast, the conjugates prepared by solution-phase reaction (series K) show broad peaks reflecting a heterogeneous size distribution. Traces indicate large average conjugate size with 15 min reaction and some growth to larger size with 60 min reaction. With longer reaction times, the shape of the peak remains the same, but the signal intensity is decreased, suggesting that the conjugate has continued to grow to sizes that do not pass through the column prefilter. Figure 2 shows representative atomic force microscopy images of defined and solution-phase conjugates. The defined conjugates are uniform in size and shape, with size increasing with the number of layers of AP, consistent with the calculated molecular masses and HPLC results. The solution-phase conjugates show extensive aggregation of the proteins increasing with reaction time, but a broad distribution of sizes for all reaction times. Figure 3A shows results of an ELISA for TSH using the defined conjugates. When the molar concentration (based on absorbance at 565 nm of the RPE core) is held constant at 40 pM, and the antiTSH Ab is constant at about 5.7 per assembly, Vmax for the hydrolysis of PNPP increases with AP content at each TSH level. Because the molecular masses of the conjugates vary over nearly an order of magnitude, the same experiment was performed with the weight-based conjugate concentration held constant at 200 ng/mL (results shown in Figure 3B). Here, the molar concentration of conjugate A is higher by a factor of 3 and that of H is lower by a factor of 2 than their respective concentrations in Figure 3A. Most of the conjugates give signals similar to those seen with molarity-based concentrations, supporting the interpretation that the signal depends more on the AP content of each conjugate unit than on the concentration of such units present in incubation. Conjugate H shows significantly less signal in the weight-based experiment. Most likely, the high molecular mass and low molar concentra-



Figure 2. Atomic force microscopy height images of defined conjugates A, B, C, D, and H and solution conjugates K15, K60, K150, and K300 adsorbed to silicon surface. Each image is a 1 µm square. The height of features above the silicon surface is color-coded according to the scale shown.

Figure 3. Comparison of defined conjugates varying AP and Ab contents in ELISA for TSH. (A) Conjugate at 40 pM shows that the signal-generating capacity of the conjugate is dependent primarily on its AP content, and (B) conjugate at 200 ng/mL shows that the AP content dependence holds even at constant mass of conjugate (see text for explanation of discrepancy for conjugate H). Conjugates with Ab content held constant at 5.5-5.8 with varying AP: [ A (AP ) 3.5), 9 B (AP ) 10.8), 2 C (AP ) 24.8), O D (AP ) 46.9), / (AP ) 74.1). (C) Conjugate at 40 pM with AP held constant at 44.4-46.9; varying Ab shows that antibody content of conjugate has little effect on signal compared to AP content: 0 E (Ab ) 2.2), O D (Ab ) 5.8), 4 F (Ab ) 16.0), ] G (Ab ) 25.8).

tion result in slow diffusion of the conjugate to the surface containing the bound TSH. Figure 3C shows the effect of varying Ab content of the defined conjugate while holding the AP content constant at about 45 per conjugate unit. With Ab content ranging from 2.2 to 26 per conjugate unit, the signal varies only by about 20%, pointing to the AP content of each conjugate unit as the primary determinant of its ability to generate signal. DISCUSSION

In most applications, protein conjugation has been treated primarily as a combining of the desired functional properties of the proteins, for example, a specific binding property with a specific signaling property. Structural considerations are usually limited to those thought to affect these properties, such as the use of site-specific activation of the antibody to direct coupling away from the binding site. Typically, only two proteins are coupled, since a third protein would complicate the mixture. The intent of the present work is to control the physical construction of protein conjugates, placing arbitrarily chosen proteins in the desired numbers at the desired positions within the assembly.

Other subdisciplines of chemistry have achieved a high level of control of reactions by use of solid supports. Peptides (10), nucleic acids (11, 12), and saccharides (13) with the desired sequence of subunits can be built up in discrete steps from specific starting points on the solid phase and finally released by chemical treatment. Intermediate reactions in the synthesis are driven to completion by use of excess reagents that are easily washed from the support before starting the next step. In the present work, immobilization of the starting material provides the same benefits, but even more importantly it prevents the uncontrolled polymerization that the plurality of reactive sites per molecule would lead to in a solutionphase reaction. In peptide, nucleic acid, and saccharide synthesis, the target is a single chemical species, and precise control of the functional groups undergoing reaction is essential. For antibody-enzyme conjugates, linkage may occur at any of many different residues of either protein and still yield a product with the desired properties. Therefore, the target can be defined at a more macroscopic level to be a linkage of the desired protein species in the desired number and location in each independent unit. This relaxed requirement for control allows selection from a


wide range of linking agents, and permits the use of the aqueous reaction media needed to retain binding and enzymatic activity of the proteins. Many of the same considerations for affinity chromatography hold in solid-phase conjugation. The support must allow free movement of proteins through its pores, but must also provide a high surface area for binding the desired proteins. The support must also be subject to modification to attach the chemical groups intended to interact with the proteins. Cross-linked agarose meets these criteria. The Sepharose CL-2B used in this work has an exclusion limit of 40 MDa, sufficient to allow conjugation and elution of the assemblies prepared here. Periodate oxidation produces aldehyde groups, which can be further modified as needed. Several chemistries are available for linking proteins to the support and to other proteins. The linkage holding the assembly to the support must be stable throughout the reaction sequence, but must be cleavable by conditions that do not damage the desired properties of the conjugate. The hydrazone linkage we chose for this work is cleaved under mild conditions expected to have minimal effect on the protein. Other possible cleavable linking groups include disulfide (cleavable by reducing agents) and vicinal diol (cleavable by periodate). These would be less generally applicable than the hydrazone link used here, since the reagents used to cleave them would be expected also to react with disulfide and carbohydrate groups, respectively, in the protein conjugates. The stepwise approach allows assembly of multiple proteins, with assurance that each conjugate unit will contain the desired quantity of each. Here, we have taken advantage of this to include a third protein, RPE, as the core. This adds a third property to the conjugate: a specific optical absorbance allowing accurate quantitation of the product. The high extinction coefficient of RPE at wavelengths at which most proteins have no absorbance makes it easy to measure its concentration even in dilute solutions containing components that would interfere with other protein measurements. Assuming that each conjugate unit contains a single core protein, the absorbance of the conjugate at 565 nm provides a direct measure of the molar concentration of the conjugate independent of the other proteins included. As a practical note, the intense color of RPE also makes it easy to follow in the manipulations involved in solidphase conjugation. Its uptake into the support gives visual confirmation that the activation steps to this point have been successful. After cleaving the linkage to the support, the elution of conjugate is easily observed, allowing collection of the product with minimal dilution by wash buffer. For the purpose of composition and molecular mass calculations, we have assumed that reaction centers remain independent throughout the sequence and that each reaction center absorbs the same number of protein molecules in each step. Both of these assumptions must be considered ideals that are approached but may not be fully achieved in the present work. A small shoulder is present on the left side of the HPLC traces in Figure 1 for all the defined conjugates except the smallest, and is most visible for conjugates B, C, and D. While no molecular mass standards are available for proteins in the desired range, the elution times of these shoulders, each near the peak of the next larger conjugate, suggest the presence of a small proportion of dimeric forms of the conjugates. Most likely, this dimerization occurs during the assembly process when two neighboring reaction centers are bridged by the linking of an added

Russell et al.

protein to active sites on both. While it may not be possible to completely eliminate the formation of dimers, it may be possible to minimize it by decreasing the density of reaction centers on the support, or by decreasing the number of reactive groups on the proteins. The assumption that each reaction center absorbs the same number of protein molecules in each step is also an approximation. In the first addition of AP-Mal, the mean uptake is 3.6 AP-Mal per RPE-SH, suggesting that some reaction centers have absorbed 4 enzyme molecules in this step and others only 3. These discrepancies are likely to be propagated through the remaining steps, and can be expected to result in some heterogeneity of size of the final conjugates. The atomic force microscope was invented in 1986 (14) as a microscopic technique to probe surface topography at molecular resolution. Since its invention, AFM has been used to image a variety of materials including polymers (15) and biological-modified surfaces (16-18). In TappingMode AFM, a cantilever holds a tip above the surface of a material, oscillating it at its resonant frequency as it is rastered across the surface. During scanning, the resonant frequency is affected by attractive or repulsive interactions with features on the surface. The motion is measured by reflecting a laser off the back of the cantilever onto a photodiode detector, and the resulting signal is converted into a 3D image by the microscope’s electronics. In this work, we imaged a selection of conjugates prepared by both solid-phase and solution methods adsorbed to silicon surfaces. The images are consistent with the HPLC results, showing average size increasing with number of layers of protein addition for the solid-phase preparations and increasing with reaction time for the solution-phase preparations. In solid-phase conjugates A-H, the protein assemblies appear approximately spherical with most at or near a maximum size characteristic of the preparation. A few appear close enough together that they may represent dimeric forms. In contrast, the solution conjugates K15-K300 show much more heterogeneity of size and shape at all reaction times. While the average size increases with reaction time, the size distribution appears largely random, and there is no indication of a preferred shape. The ability to control the composition and structure of conjugates makes possible detailed studies of the effects of these features on the functional properties of the conjugates. We demonstrated this with a limited study comparing conjugates with different enzyme and antibody contents with regard to the signal generated in an ELISA. Subject to the assumptions discussed above, the RPE chromophore allows direct measurement of the molar concentration of each conjugate independent of the presence of bound AP and antibody. When the molar concentration and Ab content of the conjugates are held constant, the assay signal at each analyte concentration increases with increasing AP content. Over the range tested, the increase in signal was somewhat less than would be expected for full participation of each additional layer of AP: conjugate H with approximately 20 times the AP content of conjugate A shows only about 10 times the signal. This may be due to decreased accessibility of substrate to AP in the interior of the assembly, either from electrostatic repulsion or because of local depletion of the substrate by AP at the surface. We also showed that, under conditions of the ELISA, the signal shows little change when the AP is held constant and the Ab content is varied over a factor of 10. This suggests that the solid-phase process may be of particular benefit when the binding agent is in limited


supply. It should be noted, however, that these experiments do not address the kinetics of binding. The rate of diffusion of the conjugate and the rate at which it binds to immobilized analyte can be expected to show dependencies on the conjugate size and the content of binding sites. The ability to control these parameters as well as the molar concentration of the conjugate may find application in such investigations. LITERATURE CITED (1) Tijssen, P. (1985) Practice and Theory of Enzyme Immunoassays, Elsevier, Amsterdam. (2) Hermanson, G. T. (1996) Bioconjugate Techniques, Academic Press, San Diego. (3) Brinkley, M. (1992) A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconjugate Chem. 3, 2-13. (4) Means, G. E., and Feeney, R. E. (1990) Chemical modifications of proteins: history and applications. Bioconjugate Chem. 1, 2-12. (5) Pastan, I., and Kreitman, R. J. (1998) Immunotoxins for targeted cancer therapy. Adv. Drug Deliv. Rev. 31, 53-88. (6) Fisher, E. A. (1985) Affinity Chromatography: A Practical Approach (Dean, P. D. G., Johnson, W. S., and Middle, F. A., Eds.) p 46, IRL Press, Oxford. (7) Duncan, R. J., Weston, P. D., and Wrigglesworth, R. (1983) A new reagent which may be used to introduce sulfhydryl groups into proteins, and its use in the preparation of conjugates for immunoassay. Anal. Biochem. 132, 68-73. (8) Tanimori, H., Kitagawa, T., Tsunoda, T., and Tsuchiya, R. J. (1981) Enzyme immunoassay of neocarzinostatin using beta-galactosidase as label. Pharmacobiodyn. 4, 812-819.

Bioconjugate Chem., Vol. 13, No. 5, 2002 965 (9) Oi, V. T., Glazer, A. N., and Stryer, L. J. (1982) Fluorescent phycobiliprotein conjugates for analyses of cells and molecules. Cell Biol. 93, 981-986. (10) Merrifield, R. B., and Stewart, J. M. (1965) Automated peptide synthesis. Nature 207, 522-523. (11) Powers, G. J., Jones, R. L., Randall, G. A., Caruthers, M. H., van de Sande, J. H., and Khorana, H. G. (1975) Optimal strategies for the chemical and enzymatic synthesis of bihelical deoxyribonucleic acids. J. Am. Chem. Soc. 97, 875-884. (12) Caruthers, M. H. (1985) Gene synthesis machines: DNA chemistry and its uses. Science 230, 281-285. (13) Plante, O. J., Palmacci, E. R., and Seeberger, P. H. (2001) Automated solid-phase synthesis of oligosaccharides. Science 291, 1523-1527. (14) Binnig, G., Quate, C. F., and Gerber, C. (1986) Atomic Force Microscope. Phys. Rev. Lett. 56, 930-933. (15) Godovsky, Y. K., and Magonov, S. N. (2000) Atomic Force Microscopy Visualization of Morphology and Nanostructure of an Ultrathin Layer of Polyethylene during Melting and Crystallization. Langmuir 16, 3549-3552. (16) Morris, V. J., Kirby, A. R., and Gunning, A. P. (1999) Atomic Force Microscopy for Biologists, Imperial College Press, London. (17) Shlyakhtenko, L. S., Gall, A. A., Weimer, J. J., Hawn, D. D., and Lyubchenko, Y. L. (1999) Atomic Force Microscopy Imaging of DNA Covalently Immobilized on a Functionalized Mica Surface. Biophys. J. 77, 568-576. (18) Mo¨ller, C., Allen, M., Elings, V., Engel, A., and Mu¨ller, D. J. (1999) Tapping-Mode Atomic Force Microscopy Produces Faithful High-Resolution Images of Protein Surfaces. Biophys. J. 77, 1150-1158.

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Solid Phase Assembly of Defined Protein Conjugates - American

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