Mechanisms Leading to an Oriented Immobilization of Recombinant

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Bioconjugate Chem. 2001, 12, 972−979

Mechanisms Leading to an Oriented Immobilization of Recombinant Proteins Derived from the P24 Capsid of HIV-1 onto Copolymers Laure Allard,§ Vale´rie Cheynet,§ Guy Oriol,§ Laurent Ve´ron,§ Franç oise Merlier,† Ge´rald Scre´min,† Bernard Mandrand,§ Thierry Delair,*,§ and Franç ois Mallet§ Unite´ Mixte UMR 2142, CNRS-bioMe´rieux, ENS-Lyon, 46, alleé d’Italie, 69364 Lyon Ce´dex 07, France, and bioMe´rieux, Chemin de l’Orme, 69280 Marcy l’Etoile, France. Received March 28, 2001; Revised Manuscript Received July 25, 2001

To investigate the mechanism leading to an oriented immobilization of recombinant proteins onto synthetic copolymers, five genetically modified HIV-1 p24 capsid proteins (RH24, RH24A4K2, RH24R6, RH24R4K2, and RH24K6) were tested for their efficiency to covalently bind to maleic anhydride-altmethyl vinyl ether (MAMVE) and N-vinyl pyrrolidone-alt-maleic anhydride (NVPMA) copolymers. These proteins contain, at their C-termini, tags differing in cationic and/or reactive amino acids density. We demonstrated that an increase of the charge and amine density in the tag enhances the coupling yield, the most efficient tag being a six lysine one. The reactivity of the proteins depends directly on the reactivity of the tag, and this led us to conclude that the tag was the site where the covalent grafting with the polymer occurred. Thus, design of such tags provides a new efficient and versatile method allowing oriented immobilization of recombinant proteins onto copolymers.

INTRODUCTION

Immobilization of proteins has been widely studied and has found numerous applications in purification processes (affinity chromatography), diagnostic field (immunoassays, biosensors), and large-scale industrial production (bioreactors). According to the expected application, many immobilization processes of proteins are commonly used ranging from ionic interactions, adsorption, complexation, or covalent binding, but covalent immobilization is often required to obtain very stable preparations with extended active life (1). However, binding often occurs via a random attachment of the proteins to the support through several amino acid residues, leading to a partial loss of biological properties, i.e., loss of enzymatic activity for an enzyme or loss of biological recognition for immobilized antibodies or antigens (2). The advantage of oriented grafting of biologically active proteins is mainly good steric accessibility of the active binding site. Regarding protein covalent immobilization as a chemical alteration of the biomolecule, it is of great interest to make this reaction occur at a specific site of the protein that is not involved in the biorecognition process, which is called oriented binding (3). This can be achieved using specific chemistry, for instance, the chemistry of thio groups of antibody Fab fragments or aldehyde chemistry after mild oxidation of carbohydrate moieties (3). With recombinant proteins, site-directed mutagenesis allows introduction of reactive groups, such as cystein residues, at virtually any position of a protein to achieve site-specific conjugation with functional polymers (4, 5). The use of amine groups for the conjugation of a protein is a more generic method than using specific chemistry since every protein has primary amines avail* To whom correspondence should be addressed: thierry. [email protected]. § Unite ´ Mixte UMR 2142. † bioMe ´ rieux.

able for conjugation. Furthermore, it is easier to use than thiol chemistry, as this chemistry does not suffer from the unwanted oxidation of the free cysteine groups, leading to unreactive protein dimers. Finally, it is a very flexible approach, as many different reaction conditions in terms of pH, ionic strength, and temperature can lead to the formation of a stable amide bond, linking the protein to the polymer. Hershfield et al. used site-directed mutagenesis to enhance protein modification with poly(ethylene glycol) by mutating arginine to lysine (6). Controlling the site of conjugation using amine chemistry is not a trivial issue, as lysine residues are randomly distributed along the protein. Our idea was to use electrostatic interactions with a polyanionic polymer to favor and direct the chemical immobilization of lysinetagged protein onto poly(maleic anhydride-alt-methyl vinyl ether), the hexalysine tag being both positively charged and reactive toward the anhydride moieties of the copolymer. Preliminary investigations demonstrated the feasibility of this approach, as the coupling yields of the tagged protein were higher than its nontagged counterpart. Furthermore, we showed that the reaction was enhanced by electrostatic interactions between the polymer and the biomolecule (7). This paper reports on a more thorough investigation on this approach, willing to address the effects of the reactivities and of the natures of both the polymer and the tag introduced on a HIV-1 modified p24 capsid protein. To assess the impact on the efficiency of the coupling reaction of (i) the primary amines density and (ii) the charge density composing the tag, competitive experiments using peptides have been performed. Investigation of the coupling isotherms and the corresponding Scatchard plots demonstrated a positive cooperative effect of the presence of the tag, provided it bore positively charged amino acids. EXPERIMENTAL PROCEDURES

Proteins. Five HIV-1 p24 recombinant proteins were obtained from Escherichia coli from a procedure derived

10.1021/bc010042s CCC: $20.00 © 2001 American Chemical Society Published on Web 10/30/2001

Oriented Immobilization of Recombinant Proteins

Bioconjugate Chem., Vol. 12, No. 6, 2001 973

Figure 1. Sequence alignment of recombinant RH24, RH24A4K2, RH24R6, RH24R4K2, and RH24K6 proteins (derived from HIV-1 p24 strain HXB2). The polyhistidine tag (H6) used for metal chelate affinity chromatography is indicated in italic, arginine (R) and lysine (K) residues are indicated in bold letters.

from Cheynet et al. (8). They all present a six-histidine tag (H) at their N-terminal end, allowing a one-step purification by ion metal affinity chromatography (IMAC). They were termed RH24, RH24A4K2, RH24R6, RH24R4K2, and RH24K6 for recombinant protein with respectively no tag at their C-terminal end, a tag containing four alanines and two lysines (A4K2), six arginines (R6), four arginines and two lysines (R4K2), and six lysines (K6) for the last one. E. coli strain XL1 was transformed with the expression plasmids. The extracts of soluble proteins were purified on an FPLC A ¨ kta system using a Zn-NTA activated gel. Purified proteins were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE) and by size exclusion chromatography on a Shodex Protein Pack column on a Kontron HPLC (detection 280 nm). Theoretical molar masses and isoelectric points were determined with the Mac Vector software (version 6.5.3) and compared with the experimental values obtained by mass spectrometry and isoelectric focusing. The amino acid sequence for each protein is reported in Figure 1. Ten lysines and 11 arginines groups are randomly dispersed along the nontagged RH24 protein macromolecule and conserved within all five recombinant proteins. NMR (9) and crystal structure data (10, 11, 12) demonstrated that, in the 3D structure, the 10 lysine groups were accessible for grafting though displaying differents reactivities (13). Protein Characterization. Mass Spectrometry (LCESI-MS). Analyses were performed with a single quadrupole API 100 mass spectrometer, 140B pumps, and a

785A detector (Perkin-Elmer). Reversed-phase liquid chromatography was performed on a C4 column (Vydac ref 214PT5115, 5 µm particle size). Solvent A was 0.1% (v/v) formic acid in water and solvent B was 0.1% (v/v) formic acid in water/acetonitrile mixture (5:95 v/v). A gradient of 40 to 60% of B was used. Isoelectric Focusing (IEF). IEF were performed with a PhastSystem and PhastGel IEF 5-8 from Pharmacia. The separation method used for that IEF required three steps: a prefocusing step (2000 V, 2 mA, 3.5 W, 15 °C, 75 Vh), the sample application (200 V, 2 mA, 3.5 W, 15 °C, 15 Vh) and the focusing step (2000 V, 5 mA, 3.5 W, 15 °C, 510 Vh). After separation, PhastGel was colored using PhastGel Blue R, a coomassie R350 dye on the PhastSystem from Pharmacia. The different steps of the fast coomassie staining were (i) fix: 20% trichloroacetic acid, (ii) wash/destain: 30% methanol and 10% acetic acid in distilled water (3:1:6), and (iii) stain: 0.02% PhastGel Blue R solution in approximately 30% methanol and 10% acetic acid in distilled water and 0.1% (w/v) CuSO4. After development, gels were dried. Copolymers. Four polymers were used, differing in molar masses and in nature of the comonomer used with maleic anhydride (MA). Alternate copolymers were obtained with either methyl vinyl ether (MVE) [sample MAMVE67 ) 67000 g/mol and sample MAMVE20 ) 20000 g/mol] or N-vinylpyrrolidone (NVP) [sample NVPMA58 ) 58400 g/mol and sample NVPMA29 ) 29100 g/mol]. MAMVE copolymers were obtained from Polysciences Inc. (Warrington, PA). NVPMA were synthesized in the laboratory according to Veron et al (14).

974 Bioconjugate Chem., Vol. 12, No. 6, 2001

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Table 1. Design of the C-Terminal Tag of the Different Recombinant p24a RH24 RH24A4K2 RH24R6 RH24R4K2 RH24K6 a

design of the C-terminal tag

amount of cationic amino acid per tag

amount of amino groups per tag

DLV AAAAKKSVDESL RRRRRRSVDESL RRRRKKSVDESL KKKKKKSVDESL

0 2 6 (22% of total R + K) 6 (22% of total R + K) 6

0 2 (16.6% of total K) 0 2 (16.6% of total K) 6 (37.5% of total K)

The conserved region of all the five recombinant p24 proteins contains 10 lysines and 11 arginines.

Table 2. Theoretical and Experimental Molar Masses and Isoelectric Points of HIV-1 Recombinant P24 Capsid Proteins RH24 RH24A4K2 RH24R6 RH24R4K2 RH24K6 a

amino acids

theoretical MMa

LC/ESI/MS determined MMa

theoretical Ipb

IEF determined Ipb

243 252 252 252 252

26950 27794 28190 28134 28022

26961 27803 28199 and 27477 28144 28032

5.90 6.06 6.93 6.93 6.93

5.9 6.06 and 6.5 6.9 and 6.5 6.9 and 6.7 6.9

Molar mass. b Isoelectric point.

Coupling of Proteins to Copolymers. The appropriate amount of copolymers was dissolved in anhydrous dimethyl sulfoxide (DMSO from Aldrich) at 37 °C. To a protein buffer solution at the appropriate concentration was added the required volume of polymer solution in DMSO. A standard reaction consisted of 100 µg of protein and 5 µg of polymer in 100 µL of buffer. The final concentration of DMSO in the mixture was 5%. The reaction mixture was stirred for 3 h at 37 °C. Bioconjugates were stored at 4 °C. For the bioconjugates obtained with partially hydrolyzed polymers, the polymers were dissolved in DMSO containing 10% (volume percent) H2O and incubated 48 h at 37 °C before conjugation with proteins. Bioconjugate Characterization. Bioconjugates were analyzed by size exclusion chromatography using a Shodex Protein KW-803, a Kontron HPLC 422 pump, a Kontron HPLC autosampler 465, and a Kontron UV diode array detector. Purification was run in a 0.1 M phosphate buffer pH 6.8 0.5% (w/v) of sodium dodecyl sulfate (SDS) with a flow rate of 0.5 mL/min. Detection was achieved by measuring the protein absorbance at 280 nm (the polymer did not generate any signal under our experimental conditions). The ratio of the peak area corresponding to the polymer bound protein versus the sum of the two peaks corresponding to the unbound and bound protein (i.e., the total amount of protein involved in the reaction) gave the coupling yield (Y). To assess the average amount of protein molecules per polymer chain: N ) nY/n′ where n and n′, respectively, correspond to the number of protein molecules and the number of polymer chains in the reaction mixture. RESULTS

Protein Characterization. The five recombinant proteins described above differ at their C-terminal end by the introduction of a tag containing variable density of primary amines and/or cationic charges (Table 1). The nontagged recombinant RH24 protein contains 11 arginines and 10 lysines representing respectively 4.5 and 4.1% of the total amount of amino acids. After purification by IMAC on a Zn-NTA activated gel, the purity of the proteins was greater than 95% as determined on a Coomassie Blue stained SDS polyacrylamide gel. Every protein displayed the same behavior by size exclusion chromatography (HPLC) as well as in SDS polyacrylamide gel electrophoresis. Experimental determination of the molar masses by electrospray mass spectrometry was in accordance with the expected theoretical value for all recombinant proteins (Table 2). RH24R6 protein exhibited an additional peak correspond-

ing to the expected value minus 722 mass unit. Such a deletion may occur at the N-terminal of the protein and correspond to the MRGSHH sequence, representing a 723 Da sequence. Moreover, MRGSHH-deleted RH24R6 protein would exhibit a theoretical isoelectric point of 6.5 which is compatible with the experimental IEF gel pattern obtained (see below). Although such a deletion has never been observed for any six-histidine-tagged proteins manufactured in our laboratory (8, 15,16), the four remaining histidines are enough for an efficient purification (17). As the six-arginine tag remains nonaltered in the cleaved RH24R6 protein, even if a C-terminal deletion might occur (SVDESL-COOH and RSVDESL-COOH deletion would represent 649 and 805 Da, respectively), this protein preparation was used in further experiments. Theoretical isoelectric points were determined using Mac Vector software (version 6.5.3) and were compared to the experimental values. After isoelectric focusing, it appears on a Coomassie Bleu stained gel that all the five proteins displayed a band corresponding to the expected isoelectric point (Table 2). Nevertheless, RH24A4K2, RH24R4K2, and RH24R6 proteins displayed one or more unexpected bands which may be due to impurities, oxidized forms of p24 (data not shown) as previously described (18), or cleaved recombinant proteins as observed for RH24R6 by MS analysis. Protein Immobilization Conditions. In our previous paper (7), we demonstrated that the six-lysine-tagged protein reacted under a broader range of experimental conditions than the nontagged RH24 protein. Furthermore, it came out from this previous investigation that ionic strength was the physicochemical factor that had the most important impact on the course of the reaction, a reduction of the ionic strength of the coupling medium leading to an increase in coupling efficiency. Interestingly, the improvement was more important with the lysine-tagged protein. The present work aims at getting a better insight of the mechanisms involved in the formation of covalent conjugates between tagged proteins and reactive copolymers. Therefore, we investigated the effects of the natures and reactivities of both the polymer and the tag on the course of the immobilization reaction. Two different polymers were investigated whose reactivities were modified by controlling the level of hydrolysis of the functional groups prior to running the reaction. Tags of various compositions were genetically introduced in the proteins, and different reaction buffers were investigated. The results will be discussed first in term of immobilization efficiencies and, then, more in a fundamental viewpoint on the role of the tag in the covalent process.


Figure 2. Coupling yield of recombinant proteins RH24, RH24A4K2, RH24R6, RH24R4K2, and RH24K6 to MAMVE67 copolymer, 3 h at 37 °C, in 20 mM carbonate buffer pH 8.2 (empty bar), 50 mM tris pH 7.0 (grey bar), and 50 mM phosphate pH 7.8 (black bar). [protein] ) 0.95 g/L (3.56 × 10-9 mol) and [MAMVE67] ) 0.048 g/L (7.46 × 10-11 mol). Experiments were performed in triplicate. The indicated data is the mean value ( two standard deviations.

Figure 3. Coupling yield of recombinant proteins RH24, RH24A4K2, RH24R6, RH24R4K2, and RH24K6 to MAMVE67 (empty bar), MAMVE20 (clear grey bar), NVPMA58 (dark grey bar), and NVPMA29 (black bar) copolymers at 37 °C during 3 h in 50 mM phosphate buffer pH 7.8. [protein] ) 0.95 g/L (3.56 × 10-9 mol), [MAMVE67] ) 0.048 g/L (7.46 × 10-11 mol), [MAMVE20] ) 0.048 g/L (2.5 × 10-10 mol), [NVPMA58] ) 0.048 g/L (8.56 × 10-11 mol), and [NVPMA29] ) 0.048 g/L (1.71 × 10-10 mol). Experiments were performed in triplicate. The indicated data is the mean value ( two standard deviations.

The effect of the buffer on the course of the reaction is reported in Figure 2 for each of the proteins. Carbonate and phosphate buffers presented similar patterns for the five proteins, excepted for RH24 whose coupling yield was quite poor in carbonate buffer as previously obtained (7). Conversely, the Tris buffer exhibited a quite different behavior. The coupling yields obtained with the lessreactive RH24 and RH24A4K2 proteins were higher in Tris buffer than in phosphate or in carbonate buffers.


Unexpectedly, in the Tris buffer, the two proteins containing arginine in the tags (RH24R6 and RH24R4K2) precipitated on addition of the polymer solution in DMSO, leading to coupling yields close to 0%. Finally, the most reactive RH24K6 protein exhibited the best coupling yields close to 100% whatever the buffer. This result led us to conclude that the more reactive the tag on the protein, the less important the influence of the coupling medium on the coupling yield. The phosphate buffer was retained for extensive investigations of the parameters involved in protein-copolymer coupling reaction. This is, in addition, convenient as it is the protein storage buffer. Using the phosphate buffer, the effect of the nature and the molar mass of the polymer were investigated in terms of coupling yield for each of the five recombinant proteins used. In most cases, the lower molar mass polymers allowed slightly more efficient grafting reactions than the higher molar mass ones (Figure 3). As a general trend, the N-vinylpyrrolidone copolymers were slightly less reactive than the methyl vinyl ether based ones, reflecting their higher susceptibility to hydrolysis as reported by Ve´ron et al (14). Nevertheless, as already noticed for the buffer, no significant difference was obtained when coupling the most reactive protein RH24K6 to the four copolymers. The level to which their functional groups were hydrolyzed by water (Figure 4) altered the reactivities of the polymers. Preliminary experiments showed that after storing the MAMVE polymer in a 10% (v/v) water DMSO mixture for 24 h, no alteration was observed in coupling efficiency. After 96 h of storage, the coupling yields were lower than 70% for RH24K6, illustrating the loss of reactivity of the polymer due to hydrolysis of the functional groups. In Table 3, the effect of prehydrolysis of the polymers for 48 h prior to running the coupling reaction is shown. For RH24K6, prehydrolysis of the MAMVE copolymer resulted in a slight decrease in the immobilization efficiency. The observed decrease was more important for the NVPMA copolymer, as its hydrolysis rate is higher than MAMVE (14). For RH24 and RH24R6, which are less reactive than RH24K6, the effect of prehydrolysis on the course of the reaction is more strongly marked. In addition, the reproducibility of the reaction under these conditions seems poor; the more sensitive to hydrolysis the polymer, the more variations in the results were observed. For ease of comparison, these results can be reported in percent of decrease of the coupling yield using the nonhydrolyzed polymer as a reference (Table 3). For the nontagged RH24 protein, the drop in binding efficiency is quite dramatic ranging from 80 to 98%. Conversely, the binding efficiency is moderately affected for the lysine-tagged RH24K6 pro-

Figure 4. (a) Coupling reaction between recombinant protein and copolymer MAMVE and (b) hydrolysis competitive reaction.


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Table 3. Coupling Yield of Recombinant Proteins RH24, RH24R6, and RH24K6 to Non Hydrolyzed or Partially Hydrolyzed Copolymersa proteinsb

RH24

RH24R6

RH24K6

polymersc

A67

A20

N58

N29

A67

A20

N58

N29

A67

A20

N58

N29

nonhydrolyzedd hydrolyzedd decreasee

22 ( 3 4 ( 1.5 82

37 ( 7 2 ( 0.7 94

24 ( 6 0.5 ( 0.6 98

13 ( 3 0.4 ( 0.7 97

64 ( 3 10 ( 17 84

68 ( 4 19 ( 19 72

42 ( 21 13 ( 22 68

52 ( 3.5 9 ( 7.4 82

98 ( 2 81 ( 6 17

99 ( 2 82 ( 5 17

94 ( 8 74 ( 19 22

97 ( 5 62 ( 20 37

a Conjugation reaction 3 h at 37 °C, in 50 mM phosphate buffer pH 7.8. b [proteins] ) 0.95 g/L (3.56 × 10-9 mol). c A67: [MAMVE67] ) 0.048 g/L (7.46 × 10-11 mol), A20: [MAMVE20] ) 0.048 g/L (2.5 × 10-10 mol), N58: [NVPMA58] ) 0.048 g/L (8.56 × 10-11 mol) and N29: [NVPMA29] ) 0.048 g/L (1.71 × 10-10 mol). Experiments were performed in triplicate. d Coupling yield of recombinant proteins to nonhydrolyzed and hydrolyzed copolymers; the indicated datas are the mean value ( two standard deviations. e Decrease of the coupling yields obtained with hydrolyzed polymers referring to their nonhydrolyzed counterparts expressed in percentage.

tein, ranging from 17 to 37%. An intermediate situation is obtained for the six-arginine-tagged RH24R6 protein, which exhibits decrease binding efficiency ranging from 68 to 84%. As already noticed for the previous parameters, the overall reaction efficiency is moderately affected by the amount of copolymer reactive functions when the protein counterpart is highly reactive, such as RH24K6. Finally, the nature of the tag was investigated as shown in Table 1. In the six-lysine tag, the amino groups were both positively charged and potentially reactive with the anhydride groups of the polymer. Therefore, in the following experiments, the chemical reactivity and the presence of positive charges in the tag (‘physicochemical reactivity’) were investigated. Recombinant RH24A4K2 protein bears a tag in which four out of six lysines were substituted by alanine, a neutral amino acid, reducing therefore the amount of charged/reactive amino acids in the tag. In recombinant protein RH24R6, the tag is only composed of six arginine residues, still positively charged but barely capable of reacting with anhydride groups. The tag in protein RH24R4K2 is fully positively charged and bears two lysines that are the reactive amino acids of the tag. From Figures 2 and 3, it appears that the reactivity of the proteins depends on the presence in the tag of both positive charges and reactive amino groups. The least reactive tagged protein is RH24A4K2 featuring only two charged lysines in the tag whereas the most reactive one is the six-lysine-tagged RH24K6 (higher coupling yields). Between these two extreme examples, RH24R6 binds more efficiently to the polymer than RH24A4K2. Substituting two unreactive arginines of the R6 tag by lysines (RH24R4K2) allowed an increase in the reactivity of the protein toward coupling onto the polymer. These results clearly demonstrate that the reactivities of the proteins are directly related to the reactivities of the tag. Role of the Tag in the Immobilization Process. The reactive amino acids of the tag which so drastically affect the course of the reaction as demonstrated above, correspond to ca. 2% of modification in the amino acid composition of the proteins. So, to better understand how such low amino acid variations in protein composition can impact so drastically on the reactivity of the protein, we investigated both the intrinsic reactivity of the tag toward the polymer and their behaviors in competition experiments with the proteins. In Table 4 is reported the composition of the peptides used in further experiments. The peptides contain 18 amino acid residues. The 12 carboxyl-terminal amino acids perfectly mimic the Cterminal end of a protein. The amino-terminal sequence consists of a stretch of six amino acids different from the flanking sequences of the recombinant proteins. The two N-terminal acetylated (Ac-K6 and Ac-R6) peptides mimic a situation where a protein does not contain, in a close vicinity of the tag, any primary amino group available for grafting. Conversely, NH2-K6 and NH2-R6 peptides

Table 4. Amino Acid Sequences of the Peptides Used to Mimic the Tags and Their Coupling Yield to MAMVE67 Copolymer name

amino acid sequence

coupling yield %

NH2-Ct NH2-K6 Ac-K6 NH2-R6 Ac-R6

H2N-SFADTPYPWGWLLDEGYPDAE-COOH NH2-IEGRPGKKKKKKSVDESL-COOH AcNH-IEGRPGKKKKKKSVDESL-COOH NH2-IEGRPGRRRRRRSVDESL-COOH AcNH-IEGRPGRRRRRRSVDESL-COOH

n.d. 80 68 94 32

exhibit a free terminal amine accessible for grafting. A non K non R containing peptide was used as control (NH2-Ct). The intrinsic reactivities of the tags were assessed in conjugation reactions of the peptides with copolymer MAMVE67 under identical experimental conditions used for the proteins (50 mM phosphate buffer, pH 7.8). The following gradation in coupling efficiency was observed, NH2-R6 > NH2-K6 > Ac-K6 . Ac-R6, from the most to the least reactive peptide (Table 4). Comparison from acetylated versus nonacetylated peptides showed that, at least in such short peptides, the amino terminal group is a major site involved in grafting. In an NH2-terminal free reactive group context, the R6-containing peptide proves more valuable in grafting than the K6 one. Taken together, these two last results confirm our preliminary study on a model peptide reported earlier (19). From these data, it can be stated that arginine groups are as efficient as lysine groups at generating attractive electrostatic interactions with the copolymer. The lower grafting yield of the NH2-K6 peptide may be due to multiple reaction sites on the polymer because of the presence of seven potentially reactive primary amine. Conversely, NH2-R6 can only react via the terminal amino group and is so bound to the polymer via a single attachment point. Quite logically, Ac-K6 peptide was much more reactive than the Ac-R6 one, whose residual reactivity may be explained by the presence of the weak nucleophilic hydroxyl of serine, or/and by a disfavored, but still existing, reaction of arginine groups onto reactive anhydrides. The properties of these peptides were further evaluated in competition assays with the most reactive RH24K6 protein in grafting reactions with MAMVE67 copolymer. The deduced percentages of inhibition, resulting from the addition of increasing amounts of peptides to the coupling mixture, are reported in Figure 5. All curves displayed a characteristic shape with a more or less steep slope and a variable plateau value. At a peptide/protein molar ratio lower than 25, the percentage of inhibition increased with increasing amount of peptides in the coupling mixture, except for the control peptide NH2-Ct, which being uncharged, had no interference with the immobilization of RH24K6. The following gradation in competition efficiency was observed NH2-K6 > NH2-R6 g Ac-K6 . Ac-



Figure 5. Percent inhibition of the formation of bioconjugates RH24K6 to MAMVE67 as a function of the nature of the peptide during a competitive coupling reaction. [protein] ) 0.34 g/L (1.28 × 10-9 mol), [MAMVE67] ) 0.048 g/L (7.46 × 10-11 mol). Peptides: NH2-K6 (0), Ac-K6 (9), NH2-R6 (O), Ac-R6 (b), NH2Ct (×). Coupling reaction in 50 mM phosphate buffer pH 7.8, 3 h at 37 °C. Because samples precipitated when the peptide/ protein molar ratio was greater than 25/1, a plateau value was not obtained for curve NH2-R6 (O).

R6 in descending order. The same gradation is observed at molar ratio higher than 25. All curves tend toward a plateau, which was reached as early as a ratio of 50 by NH2-K6 peptide. No plateau could be obtained for the NH2-R6 peptide due to a precipitation of the conjugates when peptide/protein molar ratio was greater than 25. These data clearly show that in this reaction, the reactivity of the peptide is related to both its amine and charge density. Finally, the most reactive NH2-K6 peptide and its acetylated counterpart were used in competition assays, in the grafting reaction, with the five recombinant proteins bearing tags of variable reactivities. The deduced percentage of inhibition are reported in Figure 6. The curves displayed patterns similar to those described above. It appears that the inhibition potential of the peptide strongly depended on the nature of the tag on the protein (Figure 6). Ac-K6 peptide competed more efficiently with the least reactive proteins (RH24 and RH24A4K2) than with the most reactive ones (RH24R6, RH24R4K2, or RH24K6), as shown by the steepest slope and highest plateau value of the inhibition curves. The same inhibition pattern was obtained with NH2-K6 peptide, although with a more pronounced effect than its acetylated counterpart, due to greater reactivity of NH2K6 peptide as discussed above. These results show evidence that recombinant protein reactivity in the grafting reaction is directly correlated with the density of charges (R and K) and primary amines (K) within the C-terminal-fused tag. DISCUSSION

The molar masses and compositions of the polymers, as well as their reactivities, have moderate effects on the course of the reaction compared to the drastic ones observed with tags of differing compositions. The reactivities of the proteins were increased if the tag bore six positively charged amino acids, and to a better extent if it comprised functional groups susceptible to react with the anhydride moieties of the polymer (Figure 2, Figure 3, and Table 3). These results prompted us to investigate the effects of the reactivities of the tag on the mechanism of binding of the proteins. By definition, a reactive tag is

Figure 6. Percent inhibition of the five recombinant proteins RH24 (×), RH24A4K2 (2) RH24R6 (O), RH24R4K2 ([), and RH24K6 (0) in competitive reaction with (a) peptide Ac-K6 and (b) NH2-K6. [protein] ) 0.34 g/L (6.4 × 10-10 mol), [MAMVE67] ) 0.048 g/L (3.73 × 10-11 mol). Coupling reaction in 50 mM phosphate buffer pH 7.8, 3 h at 37 °C.

Figure 7. Immobilization isotherms of RH24 (×), RH24A4K2 (2), RH24R6 (O), RH24R4K2 ([), and RH24K6 (0) to MAMVE67 copolymer in 50 mM phosphate buffer pH 7.8, 3 h at 37 °C. Experiments were performed five times. The indicated data is the mean value ( one standard deviation. N corresponds to the number of proteins per polymer chain.

positively charged and its overall reactivity increased by increasing the amount of lysine residues in its composition. Immobilization isotherms presented in Figure 7 show that both the maximal amount of covalently bound protein (plateau value) and the affinity (slope) of the protein for the polymer are directly related to the amino acid composition of the tags (Table 5). The more reactive the tag, the steeper the slope of the isotherms and the more proteins were bound per polymer chain. The Scatchard plots presented in Figure 8, corresponding to the


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Table 5. Correlation between Physicochemical Features of Recombinant Proteins and Isotherms Curves Characteristics RH24 protein cationsa aminesa slopeb,c plateaub,c

RH24A4K2 tag

21 0 10 0 6.7 ( 3.4 18 ( 4.8

protein

RH24R6 tag

23 2 12 2 3.3 ( 0.8 19 ( 3.5

protein 27 10 18 ( 4.4 27 ( 2

RH24R4K2 tag 6 0

protein

RH24K6 tag

27 6 12 2 437 ( 81 28 ( 4.5

protein

tag

27 6 16 6 1743 ( 96 54 ( 6

a Number of cationic and amine residues in the recombinant protein. b Slope and plateau value of the isotherms curves determined with GraFit Software version 3.0. c Experiments were performed five times. The indicated data is the mean value plus one standard deviation.

Figure 8. Scatchard plots of (a) RH24 (×), RH24A4K2 (2), RH24R6 (O); (b) RH24R4K2 ([) and RH24K6 (0). Coupling reaction in 50 mM phosphate buffer pH 7.8, 3 h at 37 °C. N corresponds to the number of proteins per polymer chain.

isotherms, can be divided into two groups. The first group corresponds to the exponential decrease observed for proteins RH24 and RH24A4K2, respectively bearing no tag or a poorly reactive one. For these two proteins, the curve is concave up, corresponding to a binding process taking place with negative cooperativity (20), i.e. the already bound proteins preventing the binding of others via steric hindrance and/or electrostatic repulsive forces. Conversely, for proteins with a reactive tag, the Scatchard plots displayed a bell-shaped curve, revealing that the binding process occurred with positive cooperativity (21). The maxima of the bell-shaped curves were directly dependent on the reactivities of the tags; the more reactive, the higher the ratio and, thus, the more efficient the binding reaction. Similarly, the positions of the maxima along the X-axis respected the order of reactivities of the tags: situated at five proteins per chain for the least charged tag, the maximum is found at 30 for the most reactive one. The strong dependency of the performance of the reaction with the reactivities of the tags suggests the reaction to be mainly mediated by the tag, in a regioselective manner. In other words, the binding reaction preferentially takes place at the site of the tag, or in its close vicinity. Supposing saturation of the polymer is

obtained for 60 protein molecules bound per polymer chain (Nmax ) 60) as observed with the most reactive RH24K6, then the fractional saturation can be determined as θ ) N/Nmax. Hill’s plots (log(θ/(1 - θ) vs Ceq) of grafting data for RH24 and RH24A4K2 featured the same linearity, and the slope (Hill coefficient) was 0.49. This low value confirms the negative cooperativity observed in the Scatchard plots. Conversely, for the data obtained with the positively charged tags, no existing model could fit our experimental data despite that Langmuir, multiequilibrium, or Freunlich models were taken into account. Even ‘the multibinding sites ligand’ model, developed by Mc Ghee and von Hippel (22), was not successfully fitted with our experimental data though it actually predicts bell-shaped Scatchard plots for positive cooperativity. In fact, our data feature a deformed-bell shape with a linear increase of the ratio values up to the maximum. This linear increase can be explained by a high affinity of the protein for the polymer due to the existence of electrostatic interactions that exhausts the protein content of the medium, up to a level of pseudosaturation (the decreasing and plateau sections). After this saturation level, the binding process gets less efficient and becomes dependent on the protein concentration. In addition, the chemical reactivity is an important factor as, when the amino acids are susceptible to react with the polymer, the protein/polymer affinity is higher and more protein can be accommodated onto the macromolecular chain, because the grafting reaction takes place at the site of the tag. Interestingly, the shapes of the Scatchard plots obtained for the proteins proved similar to those reported earlier for peptides, the chemical reactivities of which were directly related to the amino acid composition of the tags. CONCLUSION

This paper points out the importance of the reactivity of the tag introduced in the protein on the efficiency of the covalent grafting. We demonstrated that the more reactive the tag, the less important the other parameters (i.e., the nature of the buffer, the nature and size of the polymer, and then the reactivity of the polymer) on the control of the covalent immobilization. This allows the use of a great variety of many experimental conditions to perform the covalent grafting, provided the tag be reactive enough. In a previous work, we had demonstrated that 50 molecules of a lysine-tagged protein could be immobilized per polymer chain. The accommodation of so many molecules on the polymer required an end-on conformation from the protein, taking into account its dimensions, the link between the polymer and the protein was supposed to take place via the tag. In the present work, we demonstrated that the reactivity of a protein depended directly on the reactivity of its tag, which confirmed our previous hypothesis that the tag was the


site where the interactions with the polymer occurred. The coupling isotherms and the Scatchard plots of the immobilization process of each of the tagged proteins pointed out that the binding of proteins bearing highly reactive tags took place with a positive cooperativity. Conversely, in absence of tag or with a poorly reactive one, the binding occurred with a negative cooperativity. Our investigations demonstrated that the immobilization of tagged recombinant proteins can occur in a regioselective manner, by taking profit of attractive electrostatic forces between the polymer and the natural macromolecule. This concept will be used in further developments for various applications such as diagnostics or for the realization of macromolecular assemblies for the delivery of bioactive substances. ACKNOWLEDGMENT

The authors are grateful to Dr. G. Gervasi for mass spectrometry analysis, and Drs. J. P. Charrier and J. M. Dugua for many helpful discussions. L.A. is thankful to Fondation Me´rieux for financial support. LITERATURE CITED (1) Cabral, J. M. S., and Kennedy, J. F. (1991) Covalent and coordination immobilization of proteins. Bioprocess. Technol. 14, 73-138. (2) Turkova, J. (1999) Oriented immobilization of biologically active proteins as a tool for revealing protein interactions and function. J. Chromatogr. B 722, 11-31. (3) Rao, S. V., Anderson, K. W., and Bachas, L. G. (1998) Oriented immobilization of proteins. Mikrochim. Acta 128, 127-143. (4) Stayton, P. S., Fischer, M. T., and Sligar, S. G. (1988) Determination of cytochrome b5 association reactions. Characterization of methemoglobin and cytochrome P-450cam binding to genetically engineered cytochrome b5. J. Biol. Chem. 263, 13544. (5) Chilkoti, A., Chen, G., Stayton, P. S., and Hoffman, A. S. (1994) Site-specific conjugation of a temperature-sensitive polymer to a genetically engineered protein. Bioconjugate Chem. 5, 504-507. (6) Hershfield, M. S., Chaffee, S., Koro-Johnson, L., Mary, A., Smith, A. A., and Short, S. A. (1991) Use of site-directed mutagenesis to enhance the epitope-sheilding effect of covalent modification of proteins with poly(ethylene glycol). Proc. Natl. Acad. Sci. U.S.A. 88, 7185-7189. (7) Ladavie`re, C., Delair, T., Domard, A., Novelli-Rousseau, A., Mandrand, B., and Mallet, F. (1998) Covalent immobilization of proteins onto (Maleic Anhydride-alt-Methyl Vinyl Ether) copolymers: enhanced immobilization of recombinant proteins. Bioconjugate Chem. 9, 655-661. (8) Cheynet, V., Verrier, B., and Mallet F. (1993) Overexpression of HIV-1 proteins in Escherichia coli by a modified expression vector and their one-step purification. Protein Express. Purif. 4, 367-372.

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Mechanisms Leading to an Oriented Immobilization of Recombinant

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