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Orientation Specific Immobilization of Organophosphorus Hydrolase on Magnetic Particles through Gene Fusion Jianquan Wang,† Dibakar Bhattacharyya,‡ and Leonidas G. Bachas*,† Department of Chemistry and Center of Membrane Sciences, University of Kentucky, Lexington, Kentucky 40506; and Department of Chemical and Materials Engineering, University of Kentucky, Lexington, Kentucky 40506 Received February 6, 2001; Revised Manuscript Received July 16, 2001
Recombinant DNA technology has been utilized to fuse an octapeptide, Asp-Tyr-Lys-Asp-Asp-Asp-AspLys (FLAG), to the C-terminus of organophosphorus hydrolase (OPH, EC 3.1.8.1), an enzyme capable of hydrolyzing organophosphate compounds, such as insecticides and nerve gas agents. The recombinant OPHFLAG was immobilized onto magnetic beads coated with protein A in the following ways: (a) site-directly through a monoclonal antibody (MAb) specific for the FLAG peptide; (b) through the MAb that was randomly tethered to the beads using glutaraldehyde; (c) randomly by cross-linking OPH-FLAG to protein-coated beads using glutaraldehyde. Kinetic studies demonstrated that the site-directly immobilized enzyme maintained the highest catalytic efficiency. The orientation specific immobilization strategy described in this article can be applied to other proteins, and therefore, it may find potential applications in the design of biosensors, biocatalysts, and bioreactors having immobilized proteins as their biorecognition elements. Introduction Organophosphorus hydrolase (OPH), also known as phosphotriesterase, hydrolyzes a broad range of organophosphates, such as certain pesticides and nerve gas agents.1 These compounds are very toxic to animals including humans because they irreversibly inhibit acetylcholinesterase. Genes encoding OPH, opd, have been cloned separately from FlaVobacterium sp. and Pseudomonas diminuta; these two genes have exactly the same sequence.2,3 The opd gene also codes for a leader sequence at the N-terminus of the enzyme OPH, which is responsible for translocating the enzyme to the periplasmic region of the corresponding organisms. When expressed in Escherichia coli, it was found that OPH could not be detected in the medium due to partial cleavage of the leader sequence.4 It was also found that deletion of the bases encoding the 29 amino acid leader sequence from the native opd gene enhanced OPH expression and stability in E. coli.3,5 Recently, the opd gene was fused to sequences coding either the E. coli Lpp-OmpA or the ice nucleation protein from Pseudomonas syringae, which allowed anchoring of OPH on the cell surface after expression in E. coli.6,7 Recently, there is increasing interest in the use of OPH both in sensors for monitoring organophosphates and in facilitating the degradation of these toxic compounds. For example, OPH-based sensors and assays have been used to determine pesticide concentrations.8-12 Caldwell and Raushel demonstrated detoxification of organophosphate pesticides * Corresponding author: Telephone: (859) 257-6350. E-mail: bachas@ pop.uky.edu. † Department of Chemistry and Center of Membrane Sciences, University of Kentucky. ‡ Department of Chemical and Materials Engineering, University of Kentucky.
using immobilized OPH.13 Russell and co-workers achieved decontamination of nerve agents using a sponge foam with cross-linked OPH.14 In all these experiments, wild type OPH was randomly immobilized to the supports. This means that the enzyme orientation is not well controlled, which, in turn, may make the active site of the enzyme not fully accessible to the substrate, leading to loss of activity.13,14 One can envision that the activity loss of an immobilized enzyme may be minimized if the enzyme molecules are immobilized in a controlled way, such that all the active sites point away from the support matrix and are readily accessible to the substrate. Several approaches are available to site-directly immobilize enzymes onto a variety of supports (see reviews in refs 15 and 16 and references therein). For example, it was previously demonstrated in our laboratory that cysteine-free subtilisin can be site-specifically attached to surfaces through a unique cysteine residue that had been introduced by sitedirected mutagenesis.17 However, this site-directed mutagenesis approach is typically limited only to those proteins that either lack or contain only one reactive amino acid residue that has a side chain functional group (e.g., thiol in cysteine). Since few proteins fit into this category, alternative approaches have to be available for the orientation specific immobilization of proteins. Affinity tags like polyhistidine have been successfully utilized in immobilized metal affinity chromatography (IMAC) and protein immobilization.18,19 Protein A and protein G have high affinity toward the Fc region of IgG and can be used to immobilize IgG antibodies.20,21 The biotin(strept)avidin interaction has also been employed to sitedirectly immobilize proteins onto (strept)avidin-coated surfaces.22-24 The gene coding for the cellulose-binding domain (CBD) of cellulose degrading enzymes can be fused
10.1021/bm015517x CCC: $20.00 © 2001 American Chemical Society Published on Web 08/23/2001
Immobilization of Organophosphorus Hydrolase
to genes of desired proteins allowing orientation specific immobilization of the resulting fusion protein to cellulose. Very recently, Richins et al. have used this approach to immobilize OPH on cellulose.25 The immobilized OPH demonstrated favorable kinetic parameters. However, there was no direct comparison between orientation specific CBD-OPH and randomly immobilized OPH in that report. In this paper, we report orientation specific immobilization of genetically modified OPH on protein A coated magnetic beads using an affinity tag - the octapeptide Asp-Try-LysAsp-Asp-Asp-Asp-Lys (FLAG). FLAG is recognized by a monoclonal antibody (MAb), which is first immobilized on the beads through immobilized protein A. The resulting beadprotein A-MAb interaction orients OPH toward the solution phase through the FLAG tag, making its active site available for hydrolysis of organophosphate compounds. Comparisons were made of the catalytic efficiency of site-directly and randomly immobilized OPH-FLAG fusion protein. Our studies show that site-directly immobilized OPH has significantly higher activity than randomly immobilized OPH. Experimental Section Reagents. Restriction enzymes and T4 DNA ligase were purchased from Gibco (Gaithersburg, MD) and Stratagene (La Jolla, CA). Phagemid vector pBS(+), E. coli JM109, BL21, and XL1-Blue cells were also obtained from Stratagene. The plasmid pJK01 harboring the wild-type OPH gene opd was kindly provided by Dr. Frank M. Raushel of Texas A & M University. Luria Bertani (LB) medium for E. coli culture was from Difco Laboratories (Detroit, MI). 5-Bromo4-chloro-3-indolyl-β-D-galactoside (X-gal), isopropyl-1-thioβ-D-galactoside (IPTG), protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and pepstatin A, anti-FLAG M2 monoclonal antibody and its affinity gel, dimethyl pimelimidate, tris(hydroxylmethyl)aminomethane (Tris), 2-(Ncyclohexylamino)ethane-sulfonic acid (CHES), and paraoxon were from Sigma (St. Louis, MO). All the buffers were prepared in deionized water obtained by using the Milli-Q water purification system (Millipore, Bedford, MA). The amount of OPH-FLAG immobilized was calculated by concentration difference of OPH-FLAG before and after immobilization determined by using the BCA kit (Pierce, Rockford, IL). Gene Fusion. To fuse the FLAG octapeptide Asp-TyrLys-Asp-Asp-Asp-Asp-Lys to the C-terminus of the OPH, the coding sequence for the peptide was incorporated into the reverse primer for the polymerase chain reaction (PCR). The forward primer had the sequence, 5′-AC GCG GAT CCG GAG GTT TAA AAT ATG TCG ATC GGC ACA GGC GAT C-3′, while the reverse primer was, 5′-ATAG GAA TTC TCA CTT ATC ATC ATC ATC CTT GTA GTC CTG CAG TGA CGC CCG CAA GGT-3′. The underlined sequences are enzyme recognition sites for BamHI, EcoRI, and PstI, respectively. The first two recognition sites facilitate positional cloning, and the PstI site helps confirm the fusion construct. The italicized sequence corresponds to the FLAG peptide, and the bolded codon is for introducing a stop codon. The opdflag gene fusion was assembled in a single PCR
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reaction. The PCR mixture (50 µL) consisted of 200 µM each of the deoxynucleotide triphosphates (dNTPs), two units of Pfu DNA polymerase, 1 µM of each of the above primers, ∼2 ng of pJK01 plasmid as the template, and the recommended polymerase reaction buffer. The PCR conditions were: 94 °C for 3 min to denature the plasmid template, 25 cycles at 94 °C for 1 min, 52 °C for 45 s, and 72 °C for 1 min, followed by incubation at 72 °C for 7 min to fully extend the partially amplified gene, then cooling the reaction mixture at 4 °C until use. The amplified gene fusion was cut with EcoRI and BamHI and cloned into the pBS(+) vector that was previously digested with the same enzymes. The resultant pOPHFLAG was transformed into XL1-Blue cells. Colonies with correct insertion appear white on LBagar plate supplemented with 100 µg/mL of ampicillin and 40 µg/mL of X-gal. Expression and Purification of OPH-FLAG Fusion Protein. To express OPH-FLAG, the confirmed plasmid pOPHFLAG was transformed into E. coli JM109 and BL21 cells. Following overnight incubation of the transformed cells on LB ampicillin plates at 37 °C, a single colony from each plate was picked up and grown overnight in a culture tube containing LB medium with 100 µg/mL of ampicillin. The overnight culture was then diluted 1:50 with fresh LBampicillin medium into a 1-L baffled flask and grown at 37 °C to an optical density of 0.8-1.0 at 600 nm (OD600). IPTG was added at a final concentration of 1 mM to the culture as an inducer, and the culture was switched to a 30 °C incubator and grown for another 24 h. After centrifugation at 5000g, 4 °C for 15 min, the cells were resuspended in TBS buffer (50 mM Tris-HCl, 150 mM NaCl, pH 7.4) and ruptured with a Fisher Scientific model 550 Sonic Dismembrator (Pittsburgh, PA). To avoid excessive bubbling and heating that may denature the target protein, the sonication was performed by a pulsation (every 10 s for 20 min) at an intermediate power level with the sample being immersed in ice. To prevent proteolysis of the fusion OPH-FLAG by proteases, a cocktail of protease inhibitors (PMSF and pepstatin A) was added to the sonication buffer. Particulate materials were removed from the supernatant containing the OPH-FLAG by first centrifugation at 10 000g for 20 min, and then by passing the clarified supernatant through a 0.2 µm syringe filter. OPH-FLAG was purified by affinity chromatography on a gel with immobilized M2 monoclonal antibody, which is specific for the FLAG peptide following manufacturer’s instructions. Wild-type OPH was expressed in JM109 cells harboring plasmid pJK01 in a fashion similar to that for OPH-FLAG. Purification of wild-type OPH was performed in a two-step process, namely, ammonium sulfate (80% w/v) precipitation of proteins from crude extract of JM109 followed by anion exchange using a BioCAD perfusion chromatography system with a Poros HQ 20 column (PerSeptive Biosystems, Framingham, MA). The protein sample was loaded to the column preequilibrated with 50 mM HEPES/MES/sodium acetate, pH 8.5 buffer. After being washed with the same buffer, OPH was eluted with a gradient elution of sodium chloride from 0.0 to 1.5 M, and fractions with high OPH activity were collected.
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Kinetic Studies. A fixed amount (0.012 µg) of OPHFLAG or wild-type OPH was added to a final volume of 1 mL of 50 mM CHES buffer, pH 9.0 containing the substrate paraoxon in concentrations ranging from 5 to 500 µM. The absorbance change at 400 nm was recorded over a period of 200 s using a HP 8543 UV-vis diode array spectrophotometer. The initial reaction rate was computed for the first 60 s. Michaelis-Menten constants Km were derived from Lineweaver-Burk plots. Orientation Specific Immobilization. Protein A coated classical magnetic beads (Bangs Laboratory, Fishers, IN) were used as the matrix for immobilization. Specifically, 100 µL of the beads suspension (equivalent to 2 mg of beads, with a surface area of 31 cm2/mg) was first washed with antibody binding buffer (50 mM sodium borate, pH 8.2). An amount of 280 µg of monoclonal antibody M2 specific for the FLAG peptide in 2 mL of the antibody binding buffer was added to these washed beads. After 1 h of incubation with gentle shaking at room temperature, the beads were washed with 100 µL of the binding buffer and recovered. Dimethyl pimelimidate (1 mg in 200 µL of 0.20 M triethanolamine, pH 8.2) was added to the beads and mixed thoroughly by vortexing. The reaction mixture was gently shaken for 1 h at room temperature. The beads were resuspended in 200 µL of 0.1 M ethanolamine, pH 8.2, for 15 min and then washed sequentially with 200 µL of 1 M NaCl, 0.10 M glycine (pH 2.8), and deionized water. OPHFLAG was diluted to a final concentration of 100 µg/mL in 50 mM sodium borate buffer (pH 8.2) and added to the washed magnetic beads. The beads were mixed by vortexing and incubated at 4 °C with gentle shaking overnight to allow the binding of OPH-FLAG by the M2 antibody. The immobilized OPH-FLAG was treated with dimethyl pimelimidate as above to covalently tether OPH-FLAG to the immobilized M2 monoclonal antibody, which had already been tethered to protein A on the beads. The beads were washed with 3 × 200 µL of borate buffer, pH 8.2, and finally resuspended in 200 µL of 50 mM borate buffer containing 0.05% (w/v) sodium azide. The immobilized OPH-FLAG was kept at 4 °C until use. Random Immobilization. For random immobilization, two different approaches were employed, both using glutaraldehyde as a cross-linker: with or without the incorporation of the monoclonal antibody between OPH-FLAG and the protein A magnetic beads. The procedure was basically the same as that for orientation specific immobilization with the following modifications. In the case where M2 MAb was used as a spacer, 500 µL of 5% (v/v) glutaraldehyde in PBS buffer (50 mM phosphate, 150 mM NaCl, pH 7.4) was added immediately after the M2 MAb had been tethered to protein A beads. The reaction mixture was incubated at 4 °C overnight. The beads were washed with 3 × 500 µL PBS buffer, and then 500 µL of 0.1 mg/mL OPH-FLAG in PBS buffer was added. The reaction proceeded at 4 °C with gentle shaking for 8 h. The beads were washed with 3 × 200 µL of PBS buffer, resuspended in 200 µL of PBS buffer with 0.05% (w/v) sodium azide and stored at 4 °C until use. In the random immobilization case when M2 MAb was omitted,
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OPH-FLAG was cross-linked directly to protein A on the beads with glutaraldehyde. Results and Discussion Construction of OPH-FLAG Fusion. Knowledge of the three-dimensional structure of enzymes facilitates the rational development of immobilization strategies that preserve enzyme activity. In the case of OPH, the enzyme is a homodimer with two identical subunits; the active sites are located away from both the N- and C-termini.26-28 The threedimensional structure of OPH also indicates that both the N- and C-termini of the enzyme are exposed, and therefore, a gene fusion approach to attach an affinity tag should be feasible at either terminus. Richins et al. successfully demonstrated the fusion of cellulose binding domain and E. coli Lpp-OmpA sequences to the N-terminus of OPH.6,25 Herein, we demonstrate that the fusion of an octapeptide to the C-terminus of OPH leads to an active enzyme that can be site-directly immobilized on surfaces. Plasmid pJK01 encoding mature OPH, which lacks the first 29 amino acid leader sequence,29was used as the template for a PCR reaction to isolate the opd gene. It has been demonstrated previously that this leader sequence deletion results in enhanced stability of the enzyme when expressed in E. coli.3,5 This mature OPH will be referred to herein as wild type (WT) OPH. The amplified opdflag fusion appeared as an intense band of ∼1.1 kb on 1.2% agarose gel electrophoresis. XL-1 blue cell colonies transformed with the plasmid DNA having the inserted opdflag were selected, and the plasmid DNA was extracted by the alkaline lysis method. Digestion of the plasmid DNA construct with BamHI and EcoRI revealed two bands on agarose gel, with one at ∼3.2 kb corresponding to the vector, and the other at 1.1 kb representing the opdflag insert. Digestion with BamHI and PstI also gave two bands at about the same positions as the BamHI and EcoRI digest. Because of positional cloning of the opdflag, all the above digestion patterns suggested that the opdflag was correctly inserted into the pBS(+) vector. Dideoxynucleotide DNA sequencing confirmed that no errors were introduced during the PCR amplification of the gene fusion opdflag. The final plasmid containing the gene fusion was designated as pOPHFLAG (Figure 1). Expression and Purification. Previous studies by other researchers indicated that lower temperature (30 instead of 37 °C) may increase the expression of wild-type OPH.30,31 Therefore, the effects of temperature and other culture conditions on the expression of OPH-FLAG were evaluated. Both JM109 and BL21 E. coli strains were found to be suitable for the expression of OPH-FLAG. One interesting observation was that high OPH activity was detected in the culture medium when JM109 cells were used as the host and when the cells were incubated at 30 °C in a culture tube for more than 1 day. OPH activity was not observed in the culture medium with BL21 cells. Any change of the above three conditions significantly reduced the OPH activity detected in the culture medium (Figure 2). The origin of the observed difference of OPH activity in the culture medium between JM109 and BL21 cells is unknown, but the
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Figure 3. Effect of pH on the activity of immobilized OPH-FLAG (9) and homogeneous OPH-FLAG (b).
Figure 1. Plasmid pOPHFLAG, which, when expressed, produces a fusion protein of organophosphorus hydrolase (OPH) with an affinity tag, the FLAG peptide, at its C-terminus.
Figure 2. Effect of culture conditions on the expression level of OPH-FLAG in the culture medium. At the indicated incubation time after induction, 5 µL of culture medium was added to a 995 µL of 50 mM CHES buffer, pH 9.0 containing 0.1 mM of paraoxon to determine enzyme activity. Activity determined as the initial rate of hydrolysis was expressed as change in absorbance per minute at 400 nm. Key: (4) 30 °C, JM109 cells; ([) 30 °C, BL21 cells; (×) 37 °C, JM109 cells; (2) 30 °C, JM109 cells without the plasmid pOPHFLAG as a control.
temperature effect may be attributed to degradation of OPH at higher temperature.31 Inclusion body formation and misfolding of the target protein at higher temperature may also contribute to the lower OPH activity observed. For large-scale purification, JM109 cells harboring pOPHFLAG (grown in 1-L flasks) were ruptured by sonication, and OPH-FLAG from the crude extract was affinity purified using an agarose gel with immobilized monoclonal antibody against the FLAG peptide. After being eluted from the affinity column with 0.10 M glycine buffer, pH 3.0, the collected fractions were neutralized with 0.20 M Tris-HCl buffer, pH 8.0. SDS-PAGE indicated that the OPH-FLAG was eluted as a major fraction. The wild-type OPH was purified by anion exchange using a BioCAD Sprint perfusion chromatography system with a POROS 50 HQ strong anionexchange column. The combined fractions having the highest OPH activity showed ∼85% purity as estimated by SDSPAGE with silver staining. Effect of pH on Hydrolysis. The enzyme-catalyzed hydrolysis profile of paraoxon as a function of pH is shown in Figure 3. The homogeneous and immobilized OPHFLAG showed similar pH profiles, both being more effective
catalysts at higher pH. As pH increases, background hydrolysis also increases but still accounts for
