Silvana Andreescu, Bogdan Bucur, and Jean-Louis Marty
The development of new enzyme immobilization techniques that will not affect catalytic activity and conformation is an important research task. Affinity tags that are present or added at a specific position far from the active site in the structure of the native proteins could be used to create strong affinity bonds between the enzyme structure and a solid support functionalized with the complementary affinity ligand. The wide diversity of affinity systems creates many possibilities for enzyme immobilization. This work describes experimental strategies for affinity immobilization of tagged enzymes onto activated supports. Protocols for attaching histidine (His) and sugar-tagged enzymes onto common supports such as commercial aminated silica beads and activated graphite-cellulose are described in detail. Such strategies are generic and could be used in any application requiring immobilized biocatalysts, making future enzyme technologies more sensitive, simpler, re-usable, and less expensive. Finally, strategies for removing nonspecific adsorption and examples of possible applications of these methods to enzyme sensors are discussed.
Key Words: Tagged enzymes; affinity immobilization; histidine; NTA; glycoenzyme; concavalin A.
In recent years, advances in immobilization techniques have remarkably influenced the design and performance of a variety of enzymatic systems ranging from bioreactors to an important number of biosensor devices. Immobilization of enzymes is of significant importance in many practical applications. In immobilized states enzymes are more stable and can also be re-used, thereby considerably reducing costs associated with immobilization. Many efforts have been focused on finding an immobilization procedure that maintains the greatest characteristics of the enzymatic system. There are several requirements for selecting the immobilization method: (1) to preserve the enzyme activity and keep it as close as pos sible to its native state, (2) to ensure stability of the biomolecule, (3) to be low cost, and (4) to have sufficient sensitivity and selectivity for the analyte of interest. In addition, an appropriate selection is dictated by the type and nature of the enzyme and/or the support, as well as the final application of the system. Various methods such as adsorption, covalent binding using bifunctional reagents (1), physical entrapment in a polymer matrix (2), self-assembled monolayers (SAM), (3) or Langmuir-Blodgett films (4) have been used to immobilize enzymes onto different surfaces. Because the overall performances of the devices based on immobilized proteins are strongly affected by this process, intensive efforts need to be maintained in order to develop successful immobilization procedures.
A new trend in enzyme immobilization is to create different (bio)affinity bonds between an "activated" support and a specific group present on the protein surface. In this case, enzyme immobilization is achieved by using an affinity tail at a specific position of the protein sequence, which does not affect the activity or the folding of the protein. A simple binding of this modified enzyme is then performed onto a support. This procedure offers several advantages including: (1) selective and oriented immobilization similar for all biomolecules because of the same connection "binding site or specific group of the support" (in the case that only an affinity tail is presented on the protein surface), (2) the minimization of the conformational changes induced in the enzyme structure, and (3) the possibility of reloading the same activated surface with enzyme and to re-use the support according to different demands. There are two main requirements to immobilize an enzyme by specific affinity interactions: (1) the choice of a biocompatible support that should either possess the necessary functional groups (with very high affinity) and be able to specifically recognize a target group present on the external surface of the protein of interest or that can be easily functionalized and (2) the availability of a specific groups on the surface of the enzyme.
The choice of the biocompatible supporting material is essential. In most cases, the native material does not meet all the needs of enzyme immobilization and therefore requires further functionalization steps. For this reason it is necessary to identify and/or to introduce different start linking groups onto the support surface (e.g., -NH2, -COOH, -OH, ...) onto which a "smart" linker can be connected. These linkers are selected to posses a high affinity for a specific group (tag) of the enzyme. They may be designed as sandwich-like structures (support-specific linker-enzyme), usually realized layer by layer in successive independent steps separated by washing of excess activation agents. This permits the optimization and tuning of the entire immobilization protocol so that the protein is strongly bound onto the support at a specific point while avoiding the denaturation processes of the enzyme. Several commercial preactivated membranes are available and can be used as supports to immobilize enzymes using this method.
Affinity tags such as histidine (His), cysteine (Cys), or mannose-binding proteins can participate in the affinity binding (5-9). However, even though many enzymes contain such residues in their amino acid sequences, their number is very limited; very few of them are located on the protein surface (5) and are accessible for binding to the support. Moreover, genetic engineering methods permit the production of tagged proteins by attaching an affinity tag to amino or carboxyl terminals far enough away from the active site to reduce the steric hindrance during the biocatalytic processes and extend the application field of this type of immobilization to enzymes lacking the required target group on their sequence (10,11).
This chapter proposes and discusses the development of two affinity approaches for the immobilization of tagged enzymes: one based on metal chelate affinity (MCA) and one based on concanavalin A (Con A) (12-15).
2.1. Acetyl Choline Esterase Immobilization Using His-Tag and Metal Chelate Affinity
1. 0.1 MPhosphate buffer solution (PBS), pH 7.0.
2. Acetyl choline esterase (AchE) from Drosophila melanogaster fGTP Technology, Toulouse, France/' wild type and recombinant AChE-(His)6. (AChE-WT is a soluble dimeric form deleted from a hydrophobic peptide at the C-terminal end from D. melanogaster AChE. AChE-(His)6 originates from AChE-WT with a 3x histidine tag replacing the loop from amino-acids 103 to 136).
3. Epibromohydrin 98% (Sigma). Caution: highly toxic and corrosive.
4. 4 MNaOH solution.
6. Metal chelate: a chemically derivative of the nitrilotriacetic acid (NTA)—the #-(5-amino-1-carboxypentyl) iminodiacetic acid—synthesized from #-benzyl-oxycarbonyl-L-lysine according to the procedure described by Hochuli et al., 1987 (6).
7. 1.5-mL Eppendorf microtubes; Pasteur pipets and bulbs.
8. Microcentrifuge (Denver Instrument Company) suitable for 1.5-mL Eppendorf microtubes to centrifuge the mixture of silica-NTA-Ni with the enzyme solution.
9. 5-mL Graduated plastic syringe.
10. Hydroxyethyl-cellulose (HEC) (Fluka).
11. Graphite powder (Timcal).
12. 0.1 M Phosphate washing buffer solution, pH 7.5, supplemented with 0.1% Tween-20.
13. 0.2 MNaCl and 2 MMgSO4 to eliminate electrostatic and hydrophobic nonspecific interactions.
14. Bovine serum albumin (fraction V, Sigma) 0.5% (w/v) prepared in PBS to saturate the adsorption binding sites of the protein.
15. Small magnetic stir bar. Magnetic stirrer types AM 3000 D (Bioblock, France) and a silicon oil bath with temperature control.
16. Buchner flask, glass filter, and suction filter pump.
2.2. Immobilization of Glycoproteins Based on Con A Affinity—Acetyl
Choline Esterase Immobilization on Aminated Silica Beads
1. 0.1 MPBS solution, pH 7.0, was supplemented with 0.1 M0.1 MCaCl2, and 0.1 M MnCl2.
2. 0.1 MCarbonate buffer solution, pH 11.0.
3. 1 mg/mL Working solutions PBS Con A extracted from Jack Bean (Canavalia ensiformis; see Note 1).
4. AChE (E.C. 220.127.116.11) type V-S Electric eel (Sigma; see Note 2).
5. Methyl a-D-mannopyranoside (Sigma).
6. Divinyl sulfone (DVS) Fluka.
7. Amino functionalized silica beads with a 25- to 40-mm diameter LiChroprep® NH2 (Merck) were used as immobilization support.
8. Acetylthiocholine chloride ATCh (Sigma) stock solution was prepared daily in water (see Note 3).
9. 1.5-mL Eppendorf microtubes were shaken with a flask shaker SF1 (Stuart®, Barloworld, Stone, UK); set at speed 4.
10. Minicentrifuge Capsule HF-120 (Tomy, Kogyo, Japan) was used to separate the beads from reaction media.
11. A vortex ReAX 2000, (Heildolph, Schwabach, Germany) was used to mix the Eppendorf microtubes during washing cycles.
3.1. Immobilization Using His-Tag and Metal Chelate Affinity
3.1.1. Functionalization of the Graphite/Hydroxyethyl-Cellulose Mixture
With the Nitriloacetic Acid-Ni and Immobilization of Acetyl Choline
1. Synthesize the NTA chelate [an NTA derivative: #-(5-amino-1-carboxypentyl) iminodiacetic] from #-benzyloxycarbonyl-L-lysine according to the procedure described by Hochuli et al. 1987; (6).
2. Check the chelation properties of the synthesized NTA and the affinity binding capacity of the AChE-(His)6 for a sepharose column loaded with NTA and complexed with Ni ions. Use the same procedure described by Hochuli et al. (1987; 6) to load the sepharose with the NTA and Ni and to equilibrate the column. A plastic syringe was packed with the sepharose-NTA-Ni.
3. Deposit 35 U AChE-(His)6 in PBS onto the sepharose column and add PBS. Collect fractions of 1.5 mL and measure the enzymatic activity in each fraction to determine the amount of the immobilized enzyme.
4. A modified procedure is used to functionalize the graphite/HEC (see Fig. 1 and Note 4) with the synthesized NTA and then with Ni (10,11) (see Note 5).
5. Activation of the hydroxyls groups of the graphite/HEC mixture: in a 10-mL glass flask add 0.2 g graphite, 0.1 g HEC, and 2 mL epibromohydrine. While stirring, add 4 mL 4% NaOH. Stir the solution for 4 h under controlled temperature conditions (30°C).
6. Wash intensively with water until a neutral pH is achieved in order to eliminate the excess of epibromohydrin and NaOH. Separate the water by centrifuge and filter.
7. Loading with the NTA derivative: in a round bottom flask containing the resulting activated graphite/HEC add 0.1 g NTA derivative, 0.2 g Na2CO3, and 1 mL distilled water. Stir the mixture overnight at 60°C. Wash with water to remove the excess of NTA. Remove the water first by centrifuge and then filter.
8. Complexation with Ni: wash with a 1% (w/v) Ni2+ solution.
9. Eliminate the excess Ni by washing with distilled water.
10. Wash with 0.1 Mphosphate washing buffer, pH 7.5, containing Tween-20, NaCl, and MgSO4 to minimize the nonspecific interactions.
11. The resulting graphite/HEC-NTA-Ni can be used for immobilization of a tagged enzyme by MCA (11). The resulting functionalized support can be employed to
construct an enzyme biosensor on which any His-tagged enzyme can be immobilized (see Fig. 2).
12. Add a solution of 0.5% (w/x) BSA to saturate the binding sites of the support accessible to enzyme adsorption.
13. Deposit a solution of His-tagged enzyme prepared in PBS, pH 7.0 (see Note 6), of the desired activity function of the final application of the enzyme system.
3.1.2. Evaluation of Nonspecific Interactions and Re-Usability of the Support
1. Perform the same procedure to immobilize a wild type nontagged enzyme (the same amount as used for the His tagged) onto a similar functionalized support.
2. Check to see if there is any enzyme bounded onto the support. This corresponds to the enzyme fixed by adsorption.
3. Immobilization of His-tagged enzymes by affinity via metal chelate is fully reversible upon addition of competitive compounds such as imidazole (Im) or upon removal of metal ion from the NTA group with stronger chelators such ethylene diamine tetraacetic acid (EDTA). Simple washing with a solution of Im or EDTA regenerates the support (see Note 7).
4. Eliminate the enzyme bound by affinity by simply washing with a 0.1 M imidazole solution (see Note 8).
5. Eliminate the Ni ions with a solution of 0.1 M EDTA (see Note 9).
3.2. Immobilization of Glycoproteins Based on Con A Affinity— Application to Acetyl Choline Esterase Immobilization on Aminated Silica Beads
The immobilization procedures were performed in a few independent steps starting with the activation of the support. The immobilization was carried out with 10 mg of silica beads used as support in 1 mL of PBS or carbonate buffers to ensure the required pH value of the reagents or biocomponents used for each step. For each phase of the protocol the reactions were performed in 1.5-mL Eppendorf microtubes, mechanically shaken for 2 h at speed 4. The removal of the reagents from the reaction media was accomplished by four washing cycles, each one con-
sisting of: reaction media centrifugation, supernatant removal, vortex stirring with 1 mL of the buffer for 1 min (see Note 10). If the buffer had to be changed between steps then the first buffer was used for the first two cycles and the new buffer in the final two washing steps.
1. Activation of the amino groups of the support was performed by stirring the beads in 1-mL carbonate buffer containing 10% DVS (v/v).
2. The activated beads were allowed to react with the carbohydrate (methyl a-D-mannopyranoside) in 1 mL of 10% DVS solution (see Fig. 3A).
3. The nonreacted DVS-activated amino groups and adsorption sites were blocked with 1 mL BSA (10% m/v in carbonate buffer).
4. 1 mg Con A dissolved in 1 mL PBS was added over the beads and the lectine was immobilized on the bounded sugar via affinity links.
5. Various enzyme quantities (0.3-3.3 U AChE) were immobilized on the support through a biorecognition mechanism.
6. In order to eliminate the nonspecific adsorption phenomena a final washing was performed after enzyme immobilization for 12 h by stirring the beads in 20 mL PBS supplemented with 1 M KCl, at 4°C (16).
7. The nonspecific AChE adsorption was evaluated by replicating the immobilization protocol, but this time the lectine was blocked with 1 mL of 10% methyl a-D-mannopyranoside in PBS before contact with enzyme (see Fig. 3B ). Also, the enzyme was dissolved in PBS which also contained 10% of methyl a-D-mannopyranoside in order to ensure continuous competition between glycoproteine and sugar for the lectine from the support.
3.3. Application of the Affinity Immobilization Methods to the Construction of a Screen-Printed Biosensor Sevice
1. The amperometric biosensors were fabricated in our laboratory using the screen-printed technology. The procedure consists of successive deposition of several layers onto a plastic support (17).
2. Then, according to each affinity immobilization procedure, a specific composite paste with or without enzyme was deposited onto the surface of the working electrode surface. For this specific application the working electrode was used as a basic support for the final immobilization of the enzyme.
3.4. Biosensor With the Enzyme Immobilized Via Affinity on the Graphite/
1. Mix the Graphite-NTA-Ni with graphite-TCNQ powder (18) (10%) and 1 mL 4% (w/v) HEC.
2. Stir the mixture for 3 h to ensure paste homogeneity.
3. Print the working paste over the working electrode surface using the screen-printed technique.
4. Store at 4°C in dry state under vacuum.
6. Deposit 2 |L of His-tagged enzyme solution at the desired activity before the use of the electrode.
7. Prepare blank electrodes with the same amount of wild enzyme to evaluate the nonspecific adsorption and to confirm the specificity of the affinity binding.
8. Rinse the electrode with the washing buffer before use.
3.5. Biosensor With the Enzyme Immobilized Via Con A
1. Mixed the aminated beads with immobilized enzyme with a paste containing 150 mg graphite-TCNQ in 1 mL 4% (w/v) HEC solution (see Note 11).
2. Deposit manually and gently spread 1|L of this paste onto the surface of working electrode.
3. Dry the electrodes for 1 h in ambient atmosphere and then for 2 h in a dessicator under a slight vacuum.
4. Store the electrodes in sealed plastic bags at +4°C (16).
1. The Con A solutions were prepared in PBS, 6 h prior to use to allow the reactivation of the denatured lectin by Ca2+ and Mn2+ (19).
2. AChE was dissolved in PBS. Small aliquots were stored until first use at -18°C and after at +4°C. AChE activity was spectrometrically (20) measured using ATCh and Ellman reagent (k = 412) each time before being used.
3. The measurement of the enzyme activity has to be carried out immediately after the addition of the substrate.
4. HEC was used as additional sources of OH groups.
5. The procedure can be adapted to any support containing OH groups exposed to the surface and able to bind to the metal chelate.
6. The pH is an important parameter for the affinity interactions and strongly influences the binding efficiency. These interactions have been shown to be stable at neutral pH (21). A pH between 4.0 and -6.0 removes the tagged proteins and this principle can be used to regenerate the support.
7. It is important to notice that these procedures regenerate only the functionalized support and not the enzyme.
8. By competition with imidazole, the enzyme fixed by affinity is removed from the support.
9. Regeneration with EDTA is less desirable since the Ni ions will elute with the enzyme and an additional use of the support involves another charging with nickel and then with the enzyme.
10. The efficiency of the washing was tested at the separation of beads with immobilized AChE from 1 mL of ATCh 1 M and no color obtained with an Ellman's reagent test.
11. The HEC solution was prepared by stirring for few hours in distilled water with a magnetic bar until a homogeneous, viscous solution was obtained. The appropriate quantity of graphite-TCNQ was added to the resulting HEC, followed by stirring for a few additional hours. The mixture was kept at +4°C.
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