ARTICLE pubs.acs.org/Biomac
Glyoxyl-Disulfide Agarose: A Tailor-Made Support for Site-Directed Rigidification of Proteins Cesar A. Godoy,† Blanca de las Rivas,‡ Valeria Grazu,†,§ Tamara Montes,† Jose Manuel Guisan,*,† and Fernando Lopez-Gallego*,† †
Departamento de Biocatalisis, Instituto de Catalisis y Petroleoquímica-CSIC, Campus UAM, Cantoblanco, 28049 Madrid, Spain Departamento de Microbiologia, Instituto de Fermentaciones Industriales, CSIC, C/Juan de la Cierva 3, 28006 CSIC, Madrid, Spain § Grupo de Biofuncionalizacion de nanopartículas y superficies (bioNanoSurf), Instituto Universitario de Nanociencia de Aragon, Campus Río Ebro, Edificio IþD, Mariano Esquillos, s/n, 50018, Zaragoza, Spain ‡
bS Supporting Information ABSTRACT: A new strategy has been developed for site-directed immobilization/rigidification of genetically modified enzymes through multipoint covalent attachment on bifunctional disulfide-glyoxyl supports. Here the mechanism is described as a two-step immobilization/ rigidification protocol where the enzyme is directly immobilized by thiol-disulfide exchange between the β-thiol of the single genetically introduced cysteine and the few disulfide groups presented on the support surface (3 μmol/g). Afterward, the enzyme is uniquely rigidified by multipoint covalent attachment (MCA) between the lysine residues in the vicinity of the introduced cysteine and the many glyoxyl groups (220 μmol/g) on the support surface. Both site-directed immobilization and rigidification have been possible only on these novel bifunctional supports. In fact, this technology has made possible to elucidate the protein regions where rigidification by MCA promoted higher protein stabilizations. Hence, rigidification of vicinity of position 333 from lipase 2 from Geobacillus thermocatenulatus (BTL2) promoted a stabilization factor of 33 regarding the unipunctual sitedirected immobilized derivative. In the same context, rigidification of penicillin G acylase from E. coli (PGA) through position β201 resulted in a stabilization factor of 1069. Remarkably, when PGA was site-directed rigidified through that position, it presented a half-life time of 140 h under 60% (v/v) of dioxane and 4 °C, meaning a derivative eight times more stable than the PGA randomly immobilized on glyoxyl-disulfide agarose. Herein we have opened a new scenario to optimize the stabilization of proteins via multipoint covalent immobilization, which may represent a breakthrough in tailor-made tridimensional rigidification of proteins.
’ INTRODUCTION Enzyme immobilization by multipoint covalent attachment (MCA) has been proven to be fruitful for increasing enzyme stability.15 Optimal MCA is achieved by supports that contain high number of reactive groups ready to react with proteins. Additionally, such reactive groups must be bound to the solid support by short spacer arms to attach tightly the enzyme to the support surface.6 In this way, the side chain of the linked residues would lose its flexibility, fixing its relative positions and thus driving to a more rigid tertiary structure that results in an increasing resistance under denaturing conditions.7 However, rigidification degree and thereby stabilization may depend on the protein region whereby MCA occurs.811 The vast majority of the immobilization protocols to achieve MCA between proteins and the solid supports are based on a nucleophilic attack of ε-amine groups from lysine residues (the most abundant nucleophiles in proteins) to electrophile groups (carboxylate, succinimidyl ester, isocyanate, epoxide, aldehyde, etc.) r 2011 American Chemical Society
on support surface. 6 Among all of these electrophiles, both epoxydes and aldehydes have been broadly used as reactive moieties to immobilize proteins covalently.1215 Nevertheless, a recent study by our group has demonstrated that glyoxyl groups drive to more intense MCA between proteins and support surfaces than epoxy ones, resulting in higher protein stabilizations.16,17 Hence, rigid and hydrophilic support like agarose matrixes activated with glyoxyl groups (named Gx) would be ideal for protein immobilization by intense MCA between enzymes and supports.18 However, this type of support limits MCA and as consequence rigidifies only the richest lysine regions because glyoxyl immobilization chemistry requires several deionized lysine residues to accomplish the immobilization.6,19 This fact may be a hurdle to achieve the highest enzyme Received: February 3, 2011 Revised: March 15, 2011 Published: March 17, 2011 1800
dx.doi.org/10.1021/bm200161f | Biomacromolecules 2011, 12, 1800–1809
Biomacromolecules stabilizations because the most unstable structural elements may not be necessarily located at the highest lysine region. For diversification of the regions whereby the immobilization takes place, a new generation of tailor-made heterofunctional supports based on the agarose matrix that promotes MCA via glyoxyl chemistry has been recently developed.16 However, this methodology does not fully control the immobilization process because the rapid absorption mechanism does not distinguish between superficial regions of the enzyme with similar physicochemical nature. This fact would mean that several regions of one single protein (e.g., regions rich in Asp and Glu) may be physicochemically complementary to the support surface. To control fully protein immobilization, many directed immobilization chemistries have been developed to attach specifically one single residue at protein surface to one single moiety on the support, assuring in this way that all biomolecules are immobilized on the support through exactly the same region. Thiol-disulfide exchange is one of the broadest reactions used for site-directed immobilization because of its high chemoselective character.2022There are many studies where proteins have been directly immobilized through a unique position,2325 but only one study combines the site-directed immobilization and further rigidification of that area.26 However, in that work, the subsequent MCA to rigidify the tertiary structure may be limited because the tailor-made support contained epoxy groups.16,17 Herein we describe the easy preparation and broad usability of a novel tailor-made heterofunctional support for site-directed immobilization/rigidification. To demonstrate the applicability of such a new approach, we have site-direct-immobilized/rigidified a survey of monocysteine variants of two enzymes, penicillin G acylase (PGA) from E. coli and the lipase 2 of Geobacillus thermocatenulatus (BTL2), on those new supports. These enzymes have a known 3D structure,2729 which would facilitate the interpretation of stability results obtained by directed rigidification.
’ EXPERIMENTAL SECTION Materials. Agarose 10BCL was purchased from Agarose Bead Technologies (Madrid, Spain). Desalting PD-10 columns were purchased from GE Healthcare (Uppsala, Sweden). Epichlorhydrine, sodium metaperyodate, sodium dodecyl sulfate (SDS), Triton X-100, 2,20 -dithiodipyridine disulfide (DTDP), sodium sulfide nonahydrate (Na2S 3 9H2O), mercapthoethanol, dithiotreitol (DTT) 1,4-dioxane, p-nitrophenyl butyrate (p-NPB), and 6-nitro-3-(phenyl acetamido) benzoic acid (NIPAB), were purchased from Sigma Chem (St. Louis, MO). Other reagents were of analytical grade. Methods. All experiments were carried out at least in triplicate and standard errors were never >5%. Activation of Agarose Beads with Disulfide and/or Glyoxyl Groups. The scheme of the activation process can be seen in Figure 1A. All of the steps during support activation were carried out under mild stirring. The washing steps were performed at least 10 times with 20 volumes of water/buffer to make sure that soluble chemicals were fully removed. Glyceryl-Epoxide Support. Agarose (10 g, 10 BCL) was suspended in 44 mL of water, 16 mL of acetone, 3.28 g of NaOH, 0.2 g of NaBH4, and 11 mL of epichlorhydrine. The suspension was incubated for 16 h and then filtered and washed with excess of water. Later on, a partial hydrolysis of epoxy groups was performed by mixing 10 g of activated support with 100 mL of H2SO4 100 mM during 2 h, yielding a support named glyceryl-epoxide agarose.
ARTICLE
Glyoxyl-Disulfide Support (Gx-DS). Glyceryl-epoxide (5 g) was mixed with 75 mL of 20 mM NaIO4 solution during 2 h at 25 °C. We calculated the number of oxidized glyceryl groups by titering of remained periodate after glyceril oxidation using potassium iodide as previously described.30 Then, the support was filtered and washed. The resulting support (glyoxylepoxyde) was mixed with 100 mL of 10 Na2S dissolved in 100 mM NaHCO3 solution at pH 10 for at least 30 min; finally, the thiolated support was filtered and washed. Such support (glyoxyl-thiol) was mixed with a 75 mL of a saturated aqueous solution of 1.5 mM 2,20 DTDP. The activation degree in this last step was calculated according to the absorbance at 343 nm resulting from the 2-thiopyridone released by the thiol-disulfide exchange.31 Disulfide Support (DS). This support was activated as described for the Gx-DS support but skipping the oxidation step, resulting in a monofunctional support containing only disulfide groups as reactive ones. Bacterial Strains, Plasmids, and Enzyme Expression. E. coli strain DH5 (laboratory stock) was used for routine cloning procedures. Overproduction of both PGA and BTL2 was carried out using BL21 (DE3) (laboratory stock). The E. coli strains were routinely cultured at 37 °C in LuriaBertani (LB) broth using ampicillin (150 μg/mL) as resistance marker for both genes. PGA was overproduced at low temperatures following a described procedure.26 BTL2 was overexpressed as follows: a preinocule was cultured at 37 °C during 18 h; such culture was used to inoculate 1 L of LB media containing ampicillin (150 μg mL1) until it reached an optical density of 0.4 at 600 nm. Then, it was induced by temperature increase to 42 °C for 20 h; finally, cells were collected by centrifugation. For both enzymes, the cells were resuspended in 30 mL of 25 mM sodium phosphate pH 7 containing 3 mM benzamidine and disrupted using a French press. The lysate was centrifuged in an SS34 rotor at 20 000 rpm for 30 min at 4 °C using a Sorvall centrifuge. BTL2 variants were purified by interfacial activation using octyl agarose as described elsewhere,32,33whereas PGA variants were purified by ionic exchange34 Site-Directed Mutagenesis of Both PGA and BTL2. PGA. The different PGA monocysteine variants (SR86C, Sβ9C, Qβ112C, Sβ201C, Aβ361C, and Qβ380C) were constructed, expressed, and purified as previously described.26,34 BTL2. All site-directed mutagenesis experiments were carried out by PCR using mutagenic primers (described in Table 1 of the Supporting Information) and further digestion with endonuclease DpnI to eliminate template DNA. A double mutant was sequentially carried out for BTL2 using the plasmid pT1BTL2 as template.35 First, C64S was created, and the resulting plasmid was used as a template to create the double mutant BTL2C64S/C295S. This plasmid was used as a template to construct additional mutations (Q39C, T93C, V187C, S195C, S236C, S333C, or T342S) using different mutagenic primers (Table 1 of the Supporting Information). The resulting mutated plasmids were validated by sequencing.
Protein Determination and SDS-PAGE Electrophoresis. Protein determination was carried out following the high sensitivity protocol of the Protein Assay Kit (Pierce BCA). The purity of the different mutants was checked on 12% (w/v) SDS-PAGE gels electrophoresis stained with Coomasie blue, as described by Laemmli.36 Enzymatic Activity Assays. Esterase activity of BTL2 variants and amidase activity of PGA variants were spectrophotometrically assayed using as substrate p-NPB and NIPAB, respectively.26,32,34 Immobilization of Enzymes. All enzymes were incubated with 25 mM DTT in 50 mM sodium phosphate at pH 8 previous to immobilization to reduce the cysteine residues. The excess of the reducing agent was removed by gel filtration in GE PD-10 desalting columns following the supplier indications. Afterward, 5UI of reduced BTL2 variants in 50 mM phosphate buffer and 0.2% Triton-X100 (w/v) at pH 7 were gently incubated with 1 g of either Gx-DS or DS supports at 25 °C. Periodically, samples of supernatants were withdrawn, and their enzyme 1801
dx.doi.org/10.1021/bm200161f |Biomacromolecules 2011, 12, 1800–1809
Biomacromolecules
ARTICLE
Figure 1. Schemes of the activation procedure to obtain disulfide (DS) and glyoxyl-disulfide (Gx-DS) supports and the proposed mechanism of sitedirected MCA immobilization of proteins. Novel bifunctional supports glyoxyl-disulfide (A). The procedure to make the disulfide-containing supports was performed as described in the Methods section: both supports contain a low concentration of disulfide groups and high concentration of glyoxyl groups. Notice that the Gx-DS support contains many aldehyde groups, whereas the DS support presents many unreactive hydroxide moieties. Mechanism of the sitedirected MCA immobilization on Gx-DS support (B). In the first step, at neutral pH, a thiol-disulfide exchange takes place between the disulfide group of the support and the β-thiolate group of the monocysteine protein. This reaction directs the immobilization by the region where the cysteine was introduced. Finally, incubation at pH 10 promotes the reaction between lysine residues in the vicinity of introduced cysteine and the glyoxyl groups on support surface that after reduction produced secondary amines, establishing thus a site-directed MCA.
activity was analyzed. Immobilized enzymes were then thoroughly washed with 50 mM sodium phosphate at pH 7.0. Enzymes site-directed immobilized on Gx-DS support at pH 7.0 were additionally incubated in 0.1 M sodium bicarbonate and Triton-X100 0,2%(w/v) at pH 10 for 24 h at 25 °C.16 Finally, NaBH4 was added to 1 mg/mL concentration, the suspension was gently stirred for 30 min. Finally, derivatives were washed with 50 mM sodium phosphate at pH 7. PGA was immobilized under the same conditions than BTL2 but Triton X-100 and further incubated only for 3 h under alkaline conditions in the presence of 100 mM sodium bicarbonate pH
10 supplemented with 25% glycerol and 100 mM of phenylacetic acid to preserve enzyme activity at those conditions. Incubation of the Immobilized Enzymes with DTT. The immobilized derivatives of all enzyme variants studied here were suspended in 50 mM sodium phosphate and 400 mM DTT (1:10 w/v) at pH 8.0 and 25 °C for 1 h. Afterward, the protein and enzyme activity of supernatant were determined. As DTT is a disulfide reducing agent, enzyme molecules covalently linked to the support only through disulfide bonds are released from the support.37 1802
dx.doi.org/10.1021/bm200161f |Biomacromolecules 2011, 12, 1800–1809
Biomacromolecules Stability Assays. Residual activity was expressed as a percentage of initial activity at a given incubation time. To compare the stability of the different biocatalysts, we determined the half-life time (t1/2) by using a nonlinear fit of residual activity versus time according to a previous model.38 Thermal Inactivation of Different Enzyme Derivatives. Different suspensions of different immobilized enzyme variants were inactivated in 50 mM sodium phosphate at pH 7 and 60 and 75 °C for PGA and BTL2, respectively. Samples were withdrawn at different times, and activity was measured using enzyme assays described above. Inactivation of Different Enzyme Derivatives Immobilized Preparations in the Presence of Organic Cosolvent. Suspensions of different PGA variant’s derivatives were inactivated in 60% (v/v) of dioxane in 25 mM sodium phosphate at pH 7 and 4 °C.26 Suspensions of BTL2 derivatives were inactivated in 80% of dioxane in 50 mM Tris-HCl buffer at 30 °C.32 Samples were withdrawn at different times, and activity was measured using enzyme assays described above.
’ RESULTS AND DISCUSSION Design and Synthesis of Novel Bifunctional Glyoxyl-Disulfide Agarose Support (Gx-DS). A novel bifunctional support was
designed aiming to control both protein immobilization and rigidification onto cross-linked agarose surfaces. These matrixes contained few disulfide groups to direct the immobilization (only one bond protein-support is enough) and many glyoxyl groups for multipoint covalent attachment (MCA) (the more attachments enzyme support, the higher rigidification). The starting material was an agarose matrix activated with epoxy groups. This matrix was incubated under controlled acid conditions to hydrolyze partially the epoxy groups to diols (glyceryl) groups. Such diols were then oxidized to aldehyde using metaperiodate as oxidizing agent. The resulting solid surface contained few remaining epoxy groups and 220 μmol of glyoxyl groups per gram of support. Finally, the few epoxy groups on that surface were activated with sodium disulfide yielding free-thiol groups on support surface that were activated with 2,20 -dithiodipyridine, resulting in 3 μmol of disulfide groups per gram of support (Figure 1A). In parallel, two control supports were made; one with only disulfide groups (DS), where MCA would not be possible,26 and the other with only glyoxyl groups (Gx), where immobilization is not site-directed and can take place only under alkaline conditions.6,19 Therefore, this matrix combines the best properties of both glyoxyl (for MCA) and disulfide (for directed immobilization) groups to guarantee the rigidification of any protein’s region, which contains at least one cysteine and several lysines. The potential of such new generation of bifunctional support would be enormously enhanced using proteins with only one exposed cysteine on their primary sequence, assuring only one orientation via that residue. To do that, molecular biology tools have been used to create monocysteine enzyme variants. Characterization of Directed Immobilization-Rigidification of Proteins on the Novel Bifunctional Gx-DS Support. A Two-Step Mechanism. Directed Immobilization (First Step) Is Mediated by the Thiol-Disulfide Exchange Forming Only One Covalent but Reversible Bond. As proof of the concept of how this novel support directs both immobilization and rigidification, two monocysteine variants for two different enzymes were made. For PGA (which is natively a free-cysteine protein), the already described monocysteine variant PGA-Aβ361C has been used as an example.26 In the case of BTL2, proceedings were not so straightforward as PGA because wild-type BTL2 contained two cysteines (C64 and C295) in its primary amino acid sequence. In this sense, the two native cysteines from BTL2 (C64 and C295S) were
ARTICLE
Table 1. Immobilization Percentage at pH 7 onto Agarose Activated with Disulfide (DS), Glyoxyl (Gx), or Both Disulfide and Glyoxyl (Gx-DS) Groupsa
enzyme t (h) BTL2 PGA
monocysteine variantb
free cysteine variantc
immobilized activity
immobilized activity
Gx
Gx-DS
DS
Gx
Gx-DS
DS
