Recombinant Staphylococcal protein A with cysteine residue for preparation of af fi nity chromatography stationary phase and immunosensor applications

O. B. Gorbatiuk1, 2, A. O. Bahmachuk1, 3, L. V. Dubey1, M. O. Usenko1, 3, D. M. Irodov1, 2, O. V. Okunev1, 2, O. M. Kostenko1, A. E. Rachkov1, V.A. Kordium1, 2 1 Institute of Molecular Biology and Genetics, NAS of Ukraine 150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680 2 State Institute of Genetic and Regenerative Medicine, NAMS of Ukraine 67, Vyshhorodska Str., Kyiv, Ukraine, 04114 3 Educational and Scientifi c Center «Institute of Biology», Taras Shevchenko National University of Kyiv 64/13, Volodymyrska Str., Kyiv, Ukraine, 01601 gorbatuyk@ukr.net


Introduction
The investigations of molecular mechanisms of the pathogen-host interaction revealed microbial proteins capable of non-immune binding with host immunoglobulins without the involvement of their antigen-binding sites.Such non-immune binding of immunoglobulins by bacterial cells through immunoglobulin-binding proteins allows the microorganisms to evade the protective factors of the host body [1].Staphylococcal Protein A (SPA) was identifi ed as the fi rst protein that binds human IgG in a nonimmune manner (its Fc-fragment) [2].Now it is one of the most extensively studied immunoglobulinbinding molecules.The SPA molecule structure includes a signaling sequence [3], IgG-binding region consisting of fi ve highly homologous domains, and a C-terminal anchoring part, which attaches the protein to the bacterial cell wall [4,5].The SPA molecules are highly resistant to the denaturing factors: they are thermostable, resistant to a wide range of pH (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12), and are not destroyed by trypsin cleavage [1].Due to the high affi nity for the Fc-fragment of a wide spectrum of immunoglobulins, SPA can be used in biotechnology and health care, in particular, in test systems for the immune diagnosis and also as the components of affi nity chromatography stationary pha se for the purifi cation of antibodies or in the treatment of patients who need the autoantibodies, immune complexes, or immunoglobulins to be removed [1,6].
Among immunological test systems, immunosensors offer a high specifi city, simple operation, easy sample preparation and high sensitivity [7].Using the real-time label-free method based on the surface plasmon resonance (SPR) spectrometry for the immunosensor development seems especially promising and attractive, because it can overcome serious drawbacks of the methods based on molecular labels applications (time-and cost consuming preparation of the labeled components, a possible infl uence of labels on interacting biomolecules, multi-step detection protocols, etc.) [8].SPR spectrometer registers the changes in the refractive index of a thin layer (~200 nm) of media adjacent to a sensor surface.The processes of biomacromolecules immobilization and interactions of immobilized components with their molecules-partners change the refractive index of this layer and evoke the SPR response in real time.However, the immobilization of antibodies on the sensor surface usually leads to lowering their antigen-binding activity.A random orientation of immobilized antibody and steric constraints are believed to be the main reasons of it.To prevent this, the intermediate layer could be created, which would include the immunoglobulin-binding proteins, e.g.Staphylococcal protein A. The SPA molecules can be physically adsorbed on the sensor surface, though the genetic engineering introduction of a cysteine residue to the protein C-terminus was shown to increase the immobilization level of SPA, its IgG-binding activity as well as the further antigen-binding activity of the antibody molecules immobilized through SPA [9].The effi cient immobilization of SPA molecules, bearing cys teine residues, on the gold sensor surface is mediated by a strong thiol-gold interaction [10].
The introduction of a cysteine residue to the Cterminus of SPA molecules opens up new opportuni-ties for the creation of IgG-specifi c stationary phase for the affi nity chromatography.The preparation of such stationary phase is usually based on non-specific chemical coupling strategies.The common problems of these methods are the random orientation and low yield of functional immobilized proteins.The recent advances in synthetic chemistry have been realized in the powerful, effi cient and facile methods of the site-specifi c protein immobilization on a polymer matrix.The site-selective methods allow an attachment of uniformly oriented proteins to the bioactive site freely accessible for further applications.The cysteine residue is highly suitable for immobilization as its thiol group readily undergoes the nucleophilic substitution reaction with electrophi lic reagents, or Michael addition to α,β-un sa tu rated carbonyls (e.g., maleimides) to form stable thioether bonds.The maleimide groups strongly favor the reaction with thiols at physiological conditions (pH 6.5-7.5)[11,12].The spherical polymeric particles of monodisperse size are the most useful matrix for many solid-phase separation and analytical processes.The thiol addition to maleimide is widely used for the preparation of peptide and oligonucleotide conjugates and arrays, biosensors, fl uorescent labeling of proteins and other biomolecules, etc. [13][14][15].Therefore, we have employed thiol-maleimide chemistry for the SPA-Cys immobilization onto spherical silica beads.
The aim of the present work is the synthesis and purifi cation of recombinant Staphylococcal protein A with cysteine residue that can be used for the preparation of affi nity chromatography stationary phase and the immunosensors development.
For expression of SPA-Cys, a modifi ed auto-in duction protocol, originally described by Studier [19], was applied as follows: E. coli BL21(DE3) cells harboring the required plasmid were incubated at 37 °C overnight in 2 ml of 2 × YT medium (17 g peptone from casein, pancreatic digest «Fluka», 10 g of yeast extract «Helicon», 5 g NaCl per 1 L of water) con-taining 50 μg/ml kanamicin and 1 % glucose.For lar ge-scale production of SPA-Cys protein, 1:1000 dilutions of the overnight culture were used to inoculate 200 ml fresh 2×YT medium containing 50 μg/ ml kanamicin, 25 mM (NH 4 ) 2 SO 4 , 50 mM KH 2 PO 4 , 50 mM Na 2 HPO 4 , 1mM MgSO 4 , 0.05 % glucose, 0.2 % α-lactose, 0.5 % glycerol.The cultures were allowed to express for 18-24 h at 37 °C before the cells were harvested by centrifugation at 3000g for 20 min.The bacteria cells were disrupted by sonication on ice with 10 mM Tris-HCl buffer solution (pH 8.0), containing 1 mM EDTA.The cell lysates were precipitated by spinning at 10000g for 20 min.The supernatant was used for protein purifi cation.The protein were analyzed by SDS-PAGE in 12 % gel [20].The aliquots of supernatants and pellets were sampled and mixed with electrophoresis loading buffer containing 7 % (w/v) SDS, 40 % (v/v) glycerol, 0.25 M Tris-HCl (pH 6.8), 0.0005 % (w/v) bromophenol blue and 100 mM DTT. Gels were stained with Coomassie Brilliant Blue R250.The protein concentrations were measured by densitometry using BSA of known concentration as a standard.

Purifi cation of SPA-Cys
The purifi cation of SPA-Cys was performed by immobilized-metal affi nity chromatography (IMAC) in the native conditions. 1 ml HiTrap chelating column («GE Healthcare», USA) loaded with Ni 2+ ions was connected to the FPLC system («Pharmacia», Sweden) and equilibrated with sodium phosphate buffer solution (pH 7.4) containing 300 mM NaCl and 10 mM imidazole with fl ow rate 0.5 ml/min.The soluble protein fractions from lysates of the induced Ro setta (DE3) cells carrying the plasmid pET24-SPA-6His-Cys were fi ltered through 0.45 μm-membrane PVDF fi lter («Millipore», USA) and were applied to the column.The column was washed with the mentioned buffer to remove non-specifi cally bound proteins, and SPA-Cys was eluted with sodium phosphate buffer solution (pH 7.4) containing 300 mM NaCl and 300 mM imidazole.The homogeneity of the purifi ed protein was analyzed by SDS-PAGE in 12 % gel [20].SPA-Cys concentration was determi ned using the value of A 280 adsorption, calculated from the amino acid sequence with Vector NTI software.For large-scale purifi cation procedure, a 26/20 XK column («GE Healthcare», USA) packed with 20 ml of Ni-NTA Superfl ow resin («Qiagen», Ger many) was used.

Preparation of the maleimide-functionalized silica beads
Aminopropyl-modifi ed matrix 2 (Fig. 1) was obtained by the reaction of spherical silica beads 1 with 3-aminopropyltriethoxysilane in 95 % ethanol and subsequent surface treatment with chlorotrimethylsilane in pyridine to block polar silanol groups, following the general procedure described for glass beads amination [21].1.0 g of aminopropyl-silica was treated with 10 ml solution of reagent 3, hydroxysuccinimide ester of 3-maleimidopropionic acid, (1.2 mmol) in dry DMF for 24 h at ambient temperature with occasional stirring.The polymer was fi ltered off, washed with DMF (3 × 3 ml), and remaining free NH 2 groups were blocked with 10 % acetic anhydride in pyridine (5 ml, 10 min).The maleimide-functionalized silica beads 4 were collected by fi ltration, extensively washed with pyridine, chloroform, methanol and diethyl ether, and dried in vacuum.

Immobilization of SPA-Cys on maleimide-functionalized silica beads
Immobilization of SPA-Cys on maleimide-functionalized silica was performed by the method described by Mallik et al. [22].Before immobilization, the maleimide-functionalized silica beads were washed five times with 1.3 M potassium phosphate buffer solution, pH 7.0.Then 5 mL solution of SPA-Cys in 1.3 M potassium phosphate buffer solution, pH 7.0 with concentration 2 mg/mL was added to 0.5 g of the maleimide-functionalized silica beads.This mixture was degassed and the reaction was carried out at 4 °C for 36 h with shaking.The obtained affi nity chromatography stationary phase was separated from the reaction mixture by centrifugation and was washed fi ve times with 1.3 M potassium phosphate buffer solution (pH 7.0).After washing we estimated that about 2.1-2.3 mg of SPA-Cys was attached to 1 g of silica.The remaining free reactive groups were blocked with 10 mM cysteine.

IgG purifi cation on SPA-Cys affi nity column
Before application, the affi nity chromatography stationary phase was washed with at least 100 column volumes of 70 mM potassium phosphate buffer solution (pH 7.4).Blood serum of immunized mice, diluted with the same buffer solution was loaded onto the chromatographic column, containing 1 ml of the equilibrated SPA-Cys-based affi nity chromatography stationary phase with the fl ow rate 0.5 ml/min.The column was washed with the same buffer solution to remove unbound material, and bound antibodies were eluted with 0.1 M Na-citrate buffer so-

SPR spectrometric analysis of protein-protein interactions
SPR analysis was performed using a measuring fl owcell of the spectrometer «Plasmon-4M» and the peristaltic pump «Ismatec» (the pump speed of ~40 μl/ min).At fi rst the measuring fl ow-cell was thoroughly washed by working buffer solution (PBS) to stabilize the SPR signal.Then a sample (usually 120 μl) was injected and incubated with the pump switched off for 30 min for protein immobilization on the sensor surface and for 10 min for interactions of immobilized components with their molecules-partners.After that the measuring fl ow-cell was washed by PBS again until a stable SPR signal is obtained.To distinguish an actual sensor response caused by the interactions between the sample and sensor surface or preliminary immobilized components, from the signal caused by random fl uctuations of the medium refractive index, it was necessary prior to measuring the SPR response to wash the fl ow-cell before and after each sample by the same buffer solution [8].

Results and Discussion
To design the ORF of recombinant protein SPA-Cys, four features were taken into consideration: 1) the signaling peptide and a C-terminal anchoring part of the natural SPA do not interact with immunoglobulins (moreover, the C-terminal hydrophobic part could cause some complications in the expression of the recombinant protein); 2) the IgG-binding region consists of fi ve highly homologous domains (E, D, A, B and C); 3) the presence of 6His-tag in the protein assures its effi cient purifi cation by immobilizedmetal affi nity chromatography; 4) there is no cysteine residue in the IgG-binding domains, and the addition of cysteine residue to the C-terminus of the recombinant protein allows its reliable immobilization on the gold sensor surface and on the maleimide-functionalized silica surface.Therefore, we excluded the sequences not required for IgG-binding (signaling and a C-terminal anchoring parts of SPA) and the designed ORF of recombinant protein SPA-Cys con-tained the sequences encoding all fi ve IgG-binding domains, 6His-tag and C-terminal cysteine (Fig. 2).The auto-induction expression method, in contrast to the more expensive IPTG induction method, does not require large volumes of culture medium and special equipment.Adtionally, prolonged cultivation time (up to 24 hours) allows obtaining high yields of recombinant protein [19].The applied cultivation conditions provided a high-level expression of target proteins (~1 g/l E. coli culture).We have shown that SPA-Cys (MW = 34.5 kDa) accumulated in bacteria is in the soluble form.SPA-Cys was obtained at the multi-milligram scale in a functionally active state with purity ~95 % by one-step IMAC (Fig. 3).The IgG-binding activity of recombinant protein was confi rmed by ELISA with rabbit IgG, and the pres- The C-terminal cysteine residue provided the oriented immobilization of SPA on maleimide-activated matrix allowing the IgG-binding sites of SPA to be exposed for the interactions with IgG.The synthesis of functionalized silica beads is shown schematically in Fig. 1.The heterobifunctional cross-linking reagent 3 was used for the surface modifi cation.This reagent contains two functional groups of different reactivity, namely, maleimide residue able to interact selectively with thiols, and hydroxysuccinimide ester reacting with amines to form an amide bond.Thus reagent 3 allows the introduction of thiol-specifi c maleimide groups into the amine-modifi ed polymer.
The matrix amination was performed using the reaction of spherical silica beads 1 with 3-aminopropyltriethoxysilane [21].The amine content in various samples of aminopropyl-functionalized silica beads 2 was in the range 100-150 μmol/g.The treatment of this polymer with the amine-reactive reagent 3 in DMF resulted in the formation of modifi ed matrix 4 containing a thiol-specifi c function on a relatively fl exible linker.The concentration of maleimide groups in the polymer sample was found to be ca.70-90 μmol/g (which corresponds to the difference of amine content before and after the reaction with 3), coupling yields up to 75 %.The reaction of maleimide-modifi ed silica with thiols, including the proteins containing free SH groups, leads to their covalent attachment to the solid matrix (product 5, Fig. 1).The immobilization of SPA-Cys on the maleimide-functionalized matrix was performed under mild conditions.The obtained affi nity chromatography stationary phase was used for one-step purifi cation of antibodies.According to the SDS-PAGE results, the purity of IgG in eluted fractions was over 95 % (Fig. 4).IgG-binding capacity of the SPA-Cys affi nity chromatography stationary phase was around 10-12 mg/ml and it was stable in the pH range from 2.0 to 11.0 and in the presence of 0.1 % detergent (TWEEN 20, TWEEN 80 or Triton X-100), 8 M Urea, 6 M Gua ni dine hydrochloride, 70 % ethanol.Thus, the combination of effi cient expression systems and an optimal method for SPA immobilization on silica beads resulted in a high capacity of the affi nity chromatography stationary phase for the IgG purifi cation.
For the SPA-Cys immobilization on a gold sensor surface of SPR spectrometer «Plasmon-4m», a sample of the purifi ed recombinant Staphylococcal protein A was injected into the measuring fl ow-cell and incubated there.It led to a signifi cant increase in the sensor signal (Fig. 5).Further prolonged wash of the fl ow-cell by PBS caused only a slight decrease in the sensor signal.It means that only a small part of the sensor signal was caused by weakly bound molecules, and the major part of SPA-Cys molecules was fi rmly immobilized on the gold sensor surface.
To check, whether the immobilized molecules of SPA-Cys retain their immunoglobulin-binding properties, human IgG was injected into the measuring fl ow-cell.The sensogram (Fig. 6) shows that the introduction of 40 μg/ml IgG causes a significant sensor response without a noteworthy decrease during prolonged PBS wash.Obviously, the SPA-Cys molecules immobilized on the gold sensor surface demonstrated a high immunoglobulin-binding activity.
At the same time, the injection of other proteins (BSA and Glucose Oxidase, 40 μg/ml) does not cause noticeable changes in the sensor response (Fig. 6).So, the created bioselective element of the SPR immunosensor based on the recombinant protein SPA-Cys demonstrated the high selectivity of sensor response.Further characterizations of this bioselective element are in progress.

Conclusions
The purifi ed recombinant Staphylococcal protein A with cysteine residue was successfully used for the preparation of the affi nity chromatography stationary phase and the formation of bioselective elements of immunosensor.The affi nity chromatography stationary phase based on SPA-Cys effi ciently purifi ed the antibodies in functionally active form from serum in one-step procedure.The bioselective element of the surface plasmon resonance immunosensor based on SPA-Cys showed high activity and selectivity.

Fig. 1 .
Fig. 1.Schematic representation of the synthesis of maleimide-modifi ed matrix and its reaction with thiols.Sil -silica beads