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1 Introduction

The spleen tyrosine kinase (Syk) is a non-receptorcytoplasmic tyrosine kinase that is widely ex-pressed in hematopoietic cells. Syk plays an impor-

tant role in linking the activated B cell receptor(BCR), high affinity IgE receptor FcεRI (mast cells,eosinophils, and basophils), and FcγRI, FcγRIIA re-ceptors (macrophages, monocytes, dendritic cells,etc.) to downstream signaling pathways. Signalingfrom the pre-BCR complex is essential for clonalexpansion, maturation, proliferation, differentia-tion, and phagocytosis in pre-B cells. Key signalingtargets of activated Syk include phospholipase C(PLC)-γ2 (activates downstream ERK and JNK),PI3-K/Akt, Btk, BLNK, and α-tubulin [1]. Duringinflammation, FcεRI and Fcγ engagement leads tothe sequential activation of Syk which manifests it-self through cell degranulation, cytokine produc-tion, release of allergic mediators, and activation ofimmune cells [2–5]. Experiments with Syk anti-sense oligonucleotides have shown to abrogateFcγRIIA mediated phagocytic signaling in mono-

Research Article

Human spleen tyrosine kinase (Syk) recombinant expressionsystems for high-throughput assays

Deepika Singh1, Reema Rani1, Resmi Rajendran1, Namrata Jit Kaur1, Abhinav Pandey1, Puneet Chopra2,Tarun Jain3, Manish Kumar Jain4, Sonam Grover4, Ranjana Arya4 and Kulvinder Singh Saini1

1 Department of Biotechnology and Bioinformatics, Ranbaxy Research Laboratories, Gurgaon, Haryana, India2 Department of Pharmacology, Ranbaxy Research Laboratories, Gurgaon, Haryana, India3 Department of Medicinal Chemistry, Ranbaxy Research Laboratories, Gurgaon, Haryana, India4 School of Biotechnology, Jawaharlal Nehru University, New Delhi, India

Spleen tyrosine kinase (Syk) is an important non-receptor tyrosine kinase and its aberrant regula-tion is associated with a variety of allergic disorders and autoimmune diseases. To identify smallmolecule inhibitors of Syk in high-throughput assays, recombinant Syk protein is needed in bulkquantity. We studied the expression of recombinant human Syk in three heterologous systems: E.coli, baculovirus expression vector system (BEVS), and the cellular slime mold Dictyostelium dis-coideum (Dd). Syk activity was higher in the BEVS as compared to the Dd expression host, where-as in E. coli, no activity was observed under our assay conditions. Purified Syk kinase domain pro-tein from BEVS showed concentration dependent inhibition with OXSI-2, a known Syk inhibitor.Molecular modeling and docking studies were performed to understand the binding mode andcritical interactions of the inhibitor with catalytic domain of Syk. The BEVS generated Syk kinasedomain showed stability upon multiple freeze-thaw cycles and exhibited significantly higher levelsof tyrosine phosphorylation at pTyr525/Tyr526 in the Syk activation loop. Based on our data, we con-clude that BEVS is the ideal host to produce an active and stable enzyme, which can be success-fully employed for screening of Syk inhibitors in a high-throughput system.

Keywords: Expression · Phosphorylation · Purification · Recombinant · Syk

Correspondence: Dr. Deepika Singh, Department of Biotechnology andBioinformatics, Ranbaxy Research Laboratories, R&D III, Sec 18,Udyog Vihar, Gurgaon 122015, Haryana, IndiaE-mail: deepika.singh@ranbaxy.comFax: +91-124-2343544

Abbreviations: BEVS, baculovirus expression vector system; CD, circulardichroism; Dd, Dictyostelium discoideum; FL, full length; FPLC, fast proteinliquid chromatography; G418, geneticin; IC50, half maximum inhibitory con-centration; KD, kinase dmain; OXSI-2, [3-(1-Methyl-1H-indol-3-yl-methyl-ene)-2-oxo-2, 3-dihydro-1H-indole-5-sulfonamide; PI, protease inhibitor;Sf21, Spodoptera frugiperda 21; Syk, Spleen tyrosine kinase

Received 9 June 2009Revised 9 October 2009Accepted 9 October 2009

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cytes [6]. As outlined above, the role of Syk in in-flammation and pathophysiologic events makes itan attractive drug target for anti-inflammatorydrug discovery efforts [7].

There is a growing need for a reliable source ofpure and stable Syk enzyme for rational drug dis-covery against this target. Purified high quality en-zyme offers the opportunity to perform highthroughput screening that is useful in identifica-tion of lead compounds for further development.Functional studies with Syk enzymes are oftencomplicated by the complexity of the enzyme struc-ture, instability of the enzyme, inter-domain inter-actions, and association with various binding part-ners in the cell [8–12]. The aim of this study was toidentify an appropriate host for producing pureand stable Syk enzyme for the screening of anti-in-flammatory Syk inhibitors.

We compared the expression, purification, andcharacterization of recombinant Syk kinase from avariety of prokaryotic and eukaryotic expressionsystems including, E. coli, the cellular slime mold—Dictyostelium discoideum (Dd), and the baculovirusexpression vector system (BEVS). E. coli offers theadvantage of producing bulk amounts of the hostprotein in a cost effective manner [13]. The single-cell eukaryote Dd is an attractive host for heterolo-gous expression as it can be grown and manipulat-ed with the same ease as bacteria or yeast. Manysignaling pathways involving protein phosphoryla-tion are conserved in Dd and Kinases as protein ki-nase C, cAMP dependent protein kinase, casein ki-nase, myosin heavy chain kinases, etc., have beenpurified from Dd [14–17]. With the development ofsuccessful Dd expression vectors, many heterolo-gous eukaryotic proteins have been successfullyexpressed in Dd [18].The BEVS is another alterna-tive expression system where proper folding andpost-translational requirements of the expressedprotein are normally observed.

Previous studies on Syk using single particleelectron microscopy have shown that the N-termi-nal domain of Syk has an inhibitory effect on thecatalytic domain [19]. We observed that deletion ofthe N-amino terminal domain in Syk-KD (kinasedomain) resulted in significant increase of enzymeactivity in the BEVS, as compared to the Syk-FL(full length) enzyme. In a cross comparison studyfor Syk activity amongst the three expression sys-tems, the highest activity was reported in the Syk-KD protein expressed in the BEVS. The purifiedenzyme was found to be functionally active, showedstability upon multiple freeze–thaw cycles and wasblocked by Syk inhibitor OXSI-2 [3-(1-methyl-1H-indol-3-yl-methylene)-2-oxo-2, 3-dihydro-1H-in-dole-5-sulfonamide]. Phosphorylation sites Tyr525/

Tyr526 in the activation loop of the Syk-KD proteinare important for activity [4, 20–23]. Western blotanalysis revealed significantly greater amounts oftotal Syk tyrosine phosphorylation as well as spe-cific Syk Tyr525/Tyr526 phosphorylation in the bac-ulovirus expressed Syk-KD protein.

This is the first report directly comparing theexpression of a tyrosine kinase (Syk-FL and Syk-KD), in three expression systems: E. coli, Dd, andBEVS. Syk-KD can be efficiently generated in itshighly active form in the BEVS as shown by ra-dioactive kinase assays. Our data thus indicatesthat functionally active Syk-KD generated in theBEVS is the best source for producing active andstable enzyme, which can be successfully scaled-upfor screening of Syk inhibitors in a high throughputsystem.

2 Materials and methods

2.1 Materials

Restriction endonucleases, Protease inhibitor (PI)cocktail, and Taq polymerases were purchasedfrom Roche (Germany). General chemicals wereobtained from Sigma Chemicals (USA). Glu-tathione agarose and Ni-NTA affinity columnswere obtained from GE Health Care (USA). Syk in-hibitor was obtained from Calbiochem (USA) andwas stored as a 10 mM stock in DMSO at −20°C.[γ33–P] ATP (LCP331) was purchased from Board ofRadiation & Isotope Technology (BRIT), India.Rabbit polyclonal anti-Syk antibody, mAb anti-pTyr antibody, anti-pTyr323Syk, and anti-pTyr525/Tyr526 Syk were purchased from Cell SignalingTechnology (USA). Chemiluminescence kit forWestern blotting was obtained from Millipore(USA) and reagents for baculovirus expressionwere from Invitrogen (USA).

2.2 Cloning and expression of Syk kinase in E. coli

For expression in E. coli, Syk-FL (GenBank acces-sion no. NM_003177; nucleotides 1-1908) and Syk-KD (nucleotide 1077–1908) fragments were PCRamplified and cloned in the BamH1/Not1 sites ofpGEX4T-2 (GE Healthcare Life Sciences) vector.Positive clones were transformed in E. coli BL 21(DE3) cells for expression. Cells were grown in250 mL of terrific broth with ampicillin (50 μg/mL).Expression was induced by adding IPTG (0.5 mMfinal) followed by overnight incubation (22°C,2500 rpm). Cell pellets were resuspended in lysisbuffer (20 mM Tris pH 7.5, 150 mM NaCl, 10% Glyc-

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erol, 0.5% Triton X 100, 1 mM EDTA, 2 mM DTT, and1 × PI) and lysed by sonication.

2.3 Cloning and expression of Syk kinase in theBEVS

pFastBacHT-B (Invitrogen) was restriction digest-ed and modified to introduce a N-terminal GST tagto generate GST-pFastBacHTb vector. Syk-FL andSyk-KD were cloned in the vector. For proteinexpression a high titer virus stock was generated(5 × 108 PFU/mL) and used for infection of Sf21insect cells as per the manufacturer’s instructions.Two hundred milliliters of log phase cells(2 × 106 cells/mL) was infected with recombinantbaculovirus and incubated for 72 h. The cells werepelleted at 1000 × g for 10 min, washed with coldPBS, and re-suspended in lysis buffer (20 mM TrispH 7.5, 100 mM NaCl, 10% glycerol, 1% NP-40, 2 mMβ-mercaptoethanol, 1× PI).The cell suspension wasincubated on ice for 30 min, followed by sonicationat 200 W (for three 20 s pulses with 20 s cooling in-tervals between pulses). For co-immunoprecipita-tion experiments, the cell lysate was incubated withanti-Syk antibody for 2 h at 4°C followed by the ad-dition of Protein A beads for 1 h. The beads werewashed thrice with PBS and resuspended in SDSloading buffer. Protein samples were run on a 10%SDS gel and analyzed by immunoblotting withanti-pTyr antibody.

2.4 Cloning, expression, and culture of Ddexpressing Syk

Human Syk kinase (FL and KD) were cloned in theDd expression vector pB17S which introduces anN-terminal His-tag. The Dd axenic AX3 cells wereelectroporated, screened, and propagated as de-scribed earlier [24]. For large-scale culture, trans-formed AX3 cells (2 × 107 cells) were inoculatedinto 1-L flask containing 300 mL HL-5 medium(14.3 g/L protease peptone, 7.15 g/L yeast extract,16 g/L glucose, 0.626 g/L Na2HPO4, and 0.485 g/LKH2PO4, pH 6.5) with 10 μg/mL G418. The flaskswere incubated at 22°C/180 rpm until the densityreached log phase (4 × 106 cells/mL). Cells werepelleted by centrifugation at 1500 × g (4°C) for10 min, washed twice, and processed in lysis buffer.Cell lysate was subjected to Ni-NTA affinity purifi-cation by FPLC.

2.5 FPLC purification of recombinant expressedhuman Syk

Supernatant of cell lysates were prepared as de-scribed and cleared by ultra-centrifugation. For re-

combinant GST tagged Syk the sample was loadedon a Glutathione agarose column. The column waspre-equilibrated with binding buffer (140 mMNaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mMKH2PO4 pH 7.3). The column was attached to aFPLC system (BioLogic DuoFlow, Bio-Rad) andwashed with five column volumes of binding bufferat a flow rate of 1 mL/min. Recombinant proteinwas eluted by adding six column volumes of elutionbuffer (50 mM Tris–HCl, 10 mM reduced glu-tathione pH 8.0). For recombinant His tagged Sykthe sample was purified using a Ni-NTA affinitycolumn (Amersham, GE Healthcare, USA) as de-scribed earlier [24]. All purified enzyme prepara-tions were pooled and exchanged in storage buffer(50 mM Tris–HCl pH 7.5, 150 mM NaCl, 0.25 DTT,20% Glycerol and 1× PI). Samples from various pu-rification steps were resolved on a 10–15%SDS–PAGE followed by Western blotting with anti-Syk or anti-pTyr antibody.

2.6 Immunofluorescence

Sf21 cells expressing Syk and stable Ax3-hSyk Ddcells were seeded in fourwell Lab-Tek chamberslides.The slides were fixed with 2% paraformalde-hyde/0.1% Triton X-100 for 20 min at room temper-ature. Following blocking with 1% BSA, the cellswere incubated with rabbit polyclonal anti-Syk(1:100) for 3 h and processed as described earlier[24].

2.7 33P in vitro ATP incorporation kinase assay

Syk kinase activity was measured in a 96 well for-mat by the ability of the enzyme to phosphorylatepoly(Glu,Tyr)4:1 substrate (prepared 100 μg/mL inPBS) in reaction buffer (containing 60 mM HEPES,pH 7.5, 5 mM MgCl2, 5 mM MnCl2, 3 μM Na3Vo4,1.25 mM DTT, 10–50 μM ATP) and 0.125 μCi/well[γ33–P] ATP (∼2000 Ci/mmol) for 30 min at 30°C.Theresulting mixture (20 μL) was aliquoted on P81 pa-per which was air-dried, washed in orthophos-phoric acid solution (3 × 5 min) and acetone(3 × 5 min). Levels of incorporated [33P] ATP wasdetermined using a Wallac 1450 MicroBeta TriLuxliquid Scintillation counter. For inhibition studies,inhibitors were pre-incubated at room tempera-ture with the enzyme for 10 min before the reactionwas started by the addition of ATP. All experimentswere performed in triplicates and IC50 values werecalculated by Graphpad Prism software.

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2.8 Circular dichroism studies

The CD spectra of Syk-FL expressed in Dd, BEVS,E. coli, and Syk-KD in BEVS and E. coli wererecorded using a Jasco-815 spectropolarimeter at-tached with a peltier unit. The spectropolarimeterwas flushed with nitrogen prior to the experiments.All spectra were recorded at 25°C in far-UV regionfrom 260 to 190 nm.Three spectra were accumulat-ed to get proper S/N. Samples were prepared in10 mM phosphate buffer at pH 7.4 with a concen-tration of 100 μg/mL.The secondary structure com-position of the different samples were deconvolut-ed with the help of CDNN program (Gerald Böhm,Martin-Luther, Universität Halle-Wittenberg, Ger-many).

2.9 Molecular modeling studies

The X-ray coordinates of the crystal structure ofSyk kinase catalytic domain complexed with stau-rosporine (1XBC) were retrieved from the PDB[25]. The SYBYL suite (SYBYL, Tripos Inc.,www.tripos.com) was utilized to prepare the com-plexes and perform docking simulations. Hydrogenatoms were added to the protein and the inhibitor.

Partial atomic charges were assigned to the Sykprotein and the inhibitor using an Amber force-field and Gasteiger–Huckel charges, respectively.An ensemble of conformations of the inhibitormolecule docked into the active site of Syk kinasewas generated using the Surflex docking algorithmdefault parameter procedure [26]. The most occu-pied docking conformation cluster was selected asthe most favorable binding mode.

3 Results

3.1 Expression and purification of recombinant Sykin E. coli and the BEVS

For expression in E. coli, Syk (FL and KD) werecloned in the pGEX4-2T vector. The N-terminalGST tag has been shown not to affect Syk kinasefunctions and phosphorylation [27, 28]. Recombi-nant Syk was FPLC purified from a Glutathioneaffinity column and peak positive fractions werepooled. Representative graphs of FPLC purifica-tion (Fig. 1A) and corresponding Western blots areshown (Fig. 1B and C). GST Syk-FL protein was ob-served as a major band migrating with an expected

Figure 1. Purification of recombinant GST Syk in E. coli and Sf21 cells. Elution peak of FPLC purified fractions from glutathione agarose affinity purificationin E. coli (A) and Sf21 cells (E). FPLC purified fractions were probed with anti-Syk antibody and purified Syk-FL (B and F) and Syk-KD (C and G) proteinwere detected by Western blotting. Sf21 cells were infected with Syk expressing baculovirus and harvested after the indicated times. Cell Lysates wereresolved on SDS–PAGE and probed with anti-Syk antibody. Significant Syk expression was detected after 48 h and peaks at 72 h (D). FT, flow through; UI,uninduced; Crude, crude lysate before affinity chromatography.

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molecular weight of 100 kDa and the GST Syk-KDprotein was detected as a major band of ∼60 kDa.Post-purification the protein yield was approxi-mately 1 mg/L. Analysis of the cell lysates revealedthat majority of the protein was insoluble and at-tempts to further increase protein solubility by al-tering induction temperature, using different E. colihost strain were unsuccessful (data not shown).

In the BEVS, Syk protein expression was de-tected in the passage 3 cells (P3). Maximum yieldsof the protein were obtained at 48–72 h post-infec-tion and yields declined thereafter (Fig. 1D). Formaximum protein recovery preliminary experi-mentation with various detergents (NP-40,Triton X100, and CHAPS) revealed the best recovery in 1%NP-40 (data not shown). This concentration of de-tergent was maintained in the lysis buffer for allsubsequent purification protocols. Representativegraph of FPLC purification on a glutathione affini-ty column is shown (Fig. 1E). The presence of Sykwas confirmed by Western blotting in the respec-tive fractions (Fig. 1F and G).The protein yield was1–10 mg/L. To further confirm the expression ofSyk, we performed immuno-localization studies ininsect cells. Expression of Syk was examined at 72h post-infection by immunofluorescence stainingwith rabbit antibodies raised against Syk kinaseand FITC-conjugated goat anti-rabbit IgG (Fig. 2).In Sf21 cells some plasma membrane staining wasobserved, but majority of the kinase localizationwas cytoplasmic. As a control, uninfected Sf21 cells

were also examined; where, no specific immuno-fluorescence was detected.

3.2 Cloning and expression of Syk in Dd

In a preliminary screening of the stable Dd trans-formed clones, recombinant His Syk-FL (band size∼72 kDa) was detected by Western blotting in wholecell lysates (Fig. 3A). Similarly, stably transformedDd clones expressing His Syk-KD (band size∼35 kDa) were screened and identified via Westernblotting (Fig. 3B). For bulk culture transformed SykFL-Pb17S-10 and Syk-KD-Pb17S-4 clones weregrown. Protein was FPLC purified in an imidazolegradient from 0 to 500 mM (Fig. 3C) with a yield of0.2–1 mg/L of purified Syk protein. Expression ofFPLC purified recombinant Syk was detected byWestern blotting and corresponding blots for Sykexpressed in Dd are shown (Fig. 3D and E). Syk-FLwas also cloned in the GST tagged vector pDXA.Protein expression and enzyme activity of Syk ex-pressed in both the vectors was comparable (datanot shown). The localization of recombinant hu-man Syk kinase in the transformed Dd cells wasdetected by immunofluorescence. Transformedcells were morphologically similar to wild-typecells. Staining with Syk specific antibodies revealedbright fluorescence in the cytoplasm of the trans-formed cells (Fig. 4).

3.3 Activity analysis of Syk-FL and Syk-KDexpressed in E. coli, Dd, and BEVS and enzymestability studies

Syk kinase protein expressed in the three differentheterologous systems was tested for functional ac-tivity in a 33P in vitro ATP incorporation kinase as-say. The assay involves the ability of the recombi-nant protein to phosphorylate a glutamic acid andtyrosine copolymer [poly(Glu,Tyr)4:1]. Equal quan-tities (100 ngs) of Syk-FL protein expressed in thethree heterologous systems were tested for activi-ty. Syk kinase expressed in Dd shows low activity (asignal of 2112 cpm) and activity was comparable toSyk expressed in baculovirus (2514 cpm).Whereas,Syk kinase expressed in E. coli exhibited negligibleactivity (Fig. 5A). Syk-KD expressed in the BEVSexhibited the highest activity which was approxi-mately 8- to 10-fold higher than the Syk-KD ex-pressed in Dd.The Syk-KD protein expressed in E.coli had minimal activity (Fig. 5B). A dose depend-ent curve for Syk activity was plotted with varyingconcentrations of the Syk enzyme expressed inBEVS (Fig. 5C). With increase in enzyme concen-tration, Syk-KD activity was linear upto 250 μg/mLand plateaued at higher enzyme concentrations.

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Figure 2. Expression of recombinant Syk kinase in baculovirus Sf21 cells.Sf21 cells expressing Syk were plated in slide chambers for immuno-fluo-rescence as described in methods and cells were observed under the fluo-rescent microscope. (A) Sf21 cells expressing Syk full length (phase imageand immunofluorescence observed after staining with anti-Syk antibody).(B) Sf21 cells expressing Syk kinase domain (phase image and immuno-fluorescence observed after staining with anti-Syk antibody).

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For Syk-FL, no increase in kinase activity was ob-served with increasing enzyme concentrations.TheSyk inhibitor OXSI-2 was found to inhibit Syk-KDprotein in a dose dependent manner and IC50 val-ue was comparable to the value reported in the lit-erature (data not shown).

Syk kinase is susceptible to inactivation duringpurification and storage [8, 11, 29]. Because the en-zyme will be used in high throughput screening ofnew chemical entities (NCEs), we checked the sta-bility of the active enzyme after storage at −80°C(Fig. 6). While one aliquot was stored at −80°C theother aliquots underwent 1–10 freeze–thaw cyclesprior to analysis.The activity of freshly purified en-zyme was considered as 100%. Functional activityof the enzyme was measured using a 33P in vitroATP incorporation kinase assay. There was no sig-nificant reduction in the overall activity or Syk in-hibitory potential across different freeze–thaw cy-cles. Further, on storage at −80°C the enzyme wasstable up to 4 months without any significant lossin its activity.

3.4 BEVS generated Syk kinase domain isphosphorylated at Tyr525/Tyr526 sites in theactivation loop

We examined phosphorylation at Syk Tyr525/Tyr526

by Western blotting and assessed the levels of Sykphosphorylation relative to total Syk content. Sykkinase (FL & KD) protein expressed in BEVS

Figure 3. Expression and purification of recombinant His Syk in Dd. SykPB17S transformants were screened by Western blotting for the expres-sion of recombinant Syk FL (A) or recombinant Syk-KD (B). The blots wereprobed with human anti-Syk antibody. Representative FPLC Ni-NTA affini-ty chromatography for recombinant Syk expressed in Dd cells (C). Syk-FL(closed squares); Syk-KD (open squares); 0–100% imidazole gradient(dotted line). Western blot of the eluted protein indicated purified Syk-FLprotein (D) and purified Syk-KD protein (E). FT, flow through; UT, un-transfected control; Crude, crude lysate before affinity chromatography.

Figure 4. Expression of recombinant Syk in Dd. (A) Wild-type untrans-formed cell, (B) Dd cells transformed with Syk-FL-Pb17S (clone-10), (C)Dd cells transformed with Syk-KD-Pb17S (clone-4) were observed underthe fluorescence microscope. Phase contrast image and immunofluores-cence staining with anti-Syk antibody are shown for the respective Ddclones.

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shows tyrosine phosphorylation (blot probed withmAb anti-pTyr antibody) and specific Syk tyrosinephosphorylation in the activation loop (blot probedwith anti-Tyr525/Tyr526Syk antibody, Fig. 7A–C andG–I). Phosphorylation in the Syk linker Tyr323 is anegative regulatory phosphorylation site and afaint band was detected in Syk-FL expressed in theBEVS (Fig. 7D) [30]. This residue is deleted in thetruncated Syk-KD protein. Syk expressed in E. coliand Dd did not show any detectable tyrosine phos-phorylation or Tyr525/Tyr526 Syk phosphorylation.

Further, on immunoprecipitation with a Syk anti-body only Syk kinase expressed in the BEVS showsphosphorylation with a pTyr antibody (Fig. 7E andF).

3.5 CD spectroscopy of Syk kinase

The secondary structure content deduced from theCD spectra in far-UV region of Syk-FL proteins ex-pressed in E. coli and BEVS are nearly same as cal-culated using the CDNN software (Fig. 8 and Table1). However, the secondary structure as evidentfrom the CD spectra in far-UV region of Syk-FL ex-pressed in Dd has more helical content comparedto E. coli and BEVS kinases (Fig. 8 and Table 1).The

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Figure 5. Comparative in vitro kinase activity for recombinant Syk. (A) Syk-FL and (B) Syk-KD expressed in three heterologous expression systemswas tested in parallel by its ability to phosphorylate poly(Glu,Tyr)4:1

polypeptide. Syk kinase activity was measured by scintillation counting as[33P] ATP incorporated into substrate peptide after background subtrac-tion. Error bars represent standard deviations of three replicates. (C) Dosedependence curve of kinase activity for Syk enzyme expressed in bac-ulovirus Sf21 cells. An increase in Syk kinase activity with increasing en-zyme concentration is seen for Syk-KD protein. Syk-FL (triangles); Syk-KD(squares).

Figure 6. Effect of multiple freeze–thaws on baculovirus expressed recom-binant Syk-KD protein. Triplicate reactions were carried out in a 33P in vitroATP incorporation kinase assay as described in Section 2. Student’s t-testwas applied using Graph Pad Prism 4.0 software.

Figure 7. Tyrosine phosphorylation in BEVS generated Syk kinase protein.Purified Syk kinase FL protein expressed in the three expression systemswas resolved by SDS–PAGE and probed with Syk (A), anti-pTyr (B), anti-pSyk (Tyr525/Tyr526) (C), and anti-pSyk (Tyr323) antibody (D). Syk FL wasimmunoprecipitated with a Syk antibody and probed with anti-GST (E)and anti-pTyrosine antibody (F). Similarly, purified Syk-KD protein fromthe three expression systems was probed with Syk (G), anti-pTyr (H), anti-pSyk (Tyr525/Tyr526) antibody (I).

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beta sheets content in E. coli and baculovirus ex-pressed full length Syk kinase is approximatelythree times more than that expressed in Dd. Inter-estingly, Syk-KD expressed in E. coli showed 99.8%helical structure which may account for inactive ki-nase domain as observed in kinase activity assays(shown above). The presence of α-helical (28.4%)and β-sheets (23.7%) in Syk-KD expressed in BEVSindicates the formation of active site in the rightconformation as measured by kinase activity as-says. The CD spectra of Syk-KD expressed in Ddcould not be taken due to impurities in the proteinsample.

3.6 Molecular modeling to predict the bindingmode of Syk inhibitor in the Syk kinase activesite using docking

We investigated the potential structural determi-nants for recognition between Syk-KD and Syk in-hibitor OXSI-2. Molecular docking was done tomap the binding mode and critical interactions of

the inhibitor in the active site of Syk kinase. TheSyk-KD consists of a β-sheet N-terminal lobe andan α-helical C-terminal lobe with an active sitesandwiched between the two. The structure of theinhibitor and its binding mode in the Syk kinase ac-tive site is shown in Fig. 9.The active site of Syk ki-nase is planar in shape and rich with hydrophobicresidues. All the conformations generated usingdocking show relatively identical binding mode.The –NH and –C=O groups of the indole-2-onemoiety make hydrogen bonding interactions in the“hinge region” of the active site with the backbone–C=O of Glu449 and –NH of Ala451, respectively.Anoxygen atom of the sulphonamide group makes ahydrogen bond with the Asp512 backbone –NHatom. The Syk inhibitor also makes close van derWaals contacts (<4 Å) with the active-site residuesVal385, Ala400, Val433, Met448, Leu453, andLeu501. The indolyl phenyl ring points into theopening of the active site and makes close contactwith Leu377 and Pro455.

Table 1. Secondary structure estimation from the CD spectra for Syk-FL and Syk-KD purified from the various expression systems

Syk kinase protein Expression system Helix (%) Beta sheet (%) Beta turn (%) Random coil (%)

Full length E. coli 22.90 38.80 19.80 33.30Full length Baculovirus 21.50 44.50 20.30 32.90Full length Dictyostelium discoideum 39.00 13.4 16.40 24.30Kinase domain E. coli 99.80 0.1 3.60 0.20Kinase domain Baculovirus 28.40 23.70 18.00 33.80

CDNN analysis software was used for the estimation of secondary structure (Applied Photophysics, U.K.; copyright© Dr. Gerald Böhm Institut für Biotechnologie,Martin-Luther Universität Halle-Wittenberg)

Figure 8. (A) Far-UV CD spectra of Syk kinase FL protein expressed in (1) Dd, (2) Baculovirus, (3) E. coli, and (B) Syk-KD protein expressed in: (1) Baculovirus, (2) E. coli. Far-UV CD spectra were measured at 25°C at pH 7.4. The protein concentration was 100 μg/mL in all the cases.

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4 Discussion

Syk kinase, a cytoplasmic tyrosine kinase, is an im-portant mediator of immunoreceptor cell signalingin B cells, macrophages, neutrophils, and mast cells.Deregulation of Syk activity is often associated withmany allergic and autoimmune disorders. Becauseof the importance of Syk in signal transductionpathways and inflammation pathophysiology, con-siderable attention has been focused on the devel-opment of potent and selective inhibitors againstthis therapeutic target. The present study was un-dertaken to express and purify an active and stableform of recombinant human Syk, which can beused for high throughput screening of Syk kinaseinhibitors. In addition, we compared the expressionof recombinant human Syk enzyme in both eu-karyotic and prokaryotic hosts, viz. E. coli, Dd, andBaculovirus.

Experiments with whole Syk kinase enzymesare often complicated by the instability of the en-zyme and its susceptibility to endogenous proteol-ysis especially during multistep-purification pro-cedures [8, 11]. For example, murine Syk is highlysusceptible to proteolytic cleavage and yields a∼40 kDa fragment [12].The yield of human Syk-FLgenerated in baculovirus is also reported to be lowand is attributed to instability of the protein [9]. To

overcome some of these artifacts, recombinant en-zymes were purified by a simple one step purifica-tion procedure and their activity determined in aradioactive kinase assay. Syk expressed in E. coliexhibited poor activity. Further, this protein wasmostly insoluble due to the formation of inclusionbodies and could not be activated by autophospho-rylation with ATP (unpublished observation). Sykexpression was detected in the Dd expression sys-tem. By SDS–PAGE analysis, purified enzymeshows a molecular mass ∼72 kDa which correlateswith the predicted molecular weight for His Syk-FL. Syk expression was confirmed by immunolo-calization in the Dd cytoplasm; however, on purifi-cation the enzyme shows poor activity.

The 3D reconstruction images of Syk-FL usingsingle particle electron microscopy studies havesuggested that like Zap-70 (member of the Syk ki-nase family of proteins), Syk acquires a closed con-formation due to an inhibitory effect of its N-ter-minal domain on the catalytic domain [19, 31].While the exact mechanism of Syk in/activation isstill unclear, it has been postulated that the N-ter-minal regulatory region of Syk (composed of twotandem SH2 domains) inhibits the kinase domainby reducing the flexibility needed for transitioningto the active conformation for catalytic activity [19].This observation led us to study the expression and

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Figures 9. Molecular modeling of the in-teraction between Syk inhibitor and Sykkinase active site. (A) Chemical structureof the Syk inhibitor. Atoms highlighted inbold are involved in the hydrogen bond-ing interaction with the Syk kinase activesite residues. (B) Binding mode of Sykinhibitor in the Syk kinase active site. Ac-tive site of Syk kinase is represented as atranslucent channel and the inhibitor inBall and Stick model. Heavy atoms otherthan carbon are labeled. Non-polar hy-drogen atoms are not shown. (C) Hydro-gen bond and van der Waals interactionsof the standard compound with the ac-tive site residues of Syk kinase. Aminoacids are represented in white color lineform and inhibitor in Ball and Stick mod-el. Hydrogen bonds are shown as boldwhite dotted lines. Hinge-region is cir-cled in gray.

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10 © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

activity of both full-length and the catalytic domainof Syk kinase. In our studies, the best activity wasobserved in the Syk-KD protein expressed in bac-ulovirus. Our results thus corroborate single parti-cle electron microscopy studies which also suggestthat Syk is potentially negatively regulated by itsN-terminal.

Phosphorylation induced conformationalchanges regulate Syk kinase activity [32, 33]. In-trinsically Syk kinase activity is 100-fold higherthan ZAP-70 with which it shares 73% sequencehomology at the amino acid level.This is attributedprimarily in its ability to autophosphorylate,whereas ZAP-70 requires phosphorylation by theSrc family of kinases for its activation [34].The cat-alytic activation of Syk kinase requires the dualphosphorylation of Tyr525/Tyr526 in the activationloop. This phosphorylation event is essential forSyk function and is a molecular marker for Sykactivity [4]. The baculovirus expressed proteinwas found to be phosphorylated on Tyr525/Tyr526

residues highlighting the importance of thesephosphorylation events in regulating Syk kinaseactivity. Syk expressed in Dd lacked total Syk tyro-sine phosphorylation as well as specific tyrosinephosphorylation at Tyr525/Tyr526. While tyrosinephosphorylation of Dd proteins has been well doc-umented, the absence of Syk tyrosine phosphory-lation may be explained by the evolutionary diver-gence of this eukaryotic host [14]. Most tyrosinekinase classes vital to fundamental cellularprocesses are shared in Dd and metazoans andrepresented as the tyrosine kinase like (TLK)group [14]. Kinases involved in inflammation suchas the Syk family have homologs present in lowermetazoans as Hydra vulgaris and Ephydatia fluvi-atilis, however, this family of kinases is absent inthe Dd kinome [14, 35]. Thus despite the simplicityof the eukaryotic Dd expression host, it is plausiblethat it failed to incorporate the necessary post-translational modifications required for functionalSyk activity.

Earlier attempts to purify Syk have also shownthat there is a dramatic loss in activity on storage at−80°C. To overcome this, the protein is often con-centrated before storage. However, this procedurehas limitations as the protein was stable up to 1month only and often concentration of the proteinwas associated with undesired precipitation andloss of activity [29]. Our freeze–thaw studies withthe Syk-KD protein indicate that there was no re-duction in the overall activity or inhibitory poten-tial. Further, the enzyme was stable up to 4 monthswithout any significant loss in its activity.

A major hurdle in understanding the activitymechanism of Syk kinase FL protein is non-avail-

ability of 3D structure. As suggested by low resolu-tion EM, Syk-FL comprises of two tandem SH2 do-mains at N-terminus that are mainly β-sheets. Inaddition, 3D structure of Syk-KD indicates thepresence of β-sheets in N-terminus and that activesite is organized between N-terminal β-sheets andC-terminal α-helical regions. In our study, the CDspectra in far-UV range suggests that both β-sheetsand α-helical regions are present in the Syk-FL ex-pressed in baculovirus that may contribute to high-er activity of the protein. However, Syk-FL ex-pressed in Dd has lower β-sheet content that mayaffect the right conformation of the protein leadingto reduced functional activity. The Syk-KD ex-pressed in E. coli and baculovirus has significantstructural divergence. The Syk-KD expressed in E.coli is only α-helical suggesting loss of proper ac-tive site conformation requiring β-sheets and,hence, minimal activity. Further insights could beachieved by solving the 3D structure of Syk-FLprotein.

Molecular modeling and docking studies wereperformed to further elucidate the mechanism ofbinding of the inhibitors in the Syk-KD. Suchanalysis, early in the discovery process not onlyaids in a better design of structure–activity rela-tionship studies, but also helps in the developmentof refined biochemical assays for NCE screening.Our studies show that the Syk inhibitor binds to theATP binding pocket of the Syk Kinase. The in-hibitor binds in a similar conformation as that ofStaurosporine in the original protein–ligand com-plex [36].The inhibitor is stabilized by the hinge re-gion interactions and hydrogen bond interactionsof the sulfonamide moiety in addition to variousvan der Waals and hydrophobic contacts with theactive-site residues. These studies provide insightinto the binding mechanism of Syk kinase in-hibitors and structure-based drug design studiescan be performed with the Syk kinase domain pro-tein to design more potent inhibitors.

The development of selective Syk kinase in-hibitors as drug targets is an active area of researchfor new drug discovery. The availability of highlypurified preparations of Syk kinase in significantquantities is crucial for the identification of selec-tive and potent Syk kinase inhibitors. Our dataclearly indicates that functionally active Syk-KDgenerated in BEVS is the best source of stable andactive enzyme, which can be successfully employedin the high throughput screening of Syk inhibitorsin a wide range of assay formats.

© 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 11

We thank Prof. Rajiv Bhat, Dean, School of Biotech-nology, Jawaharlal Nehru University for the CD dataanalysis, Dr. Roop Singh Bora, Dr. Sudhir Sahdev,and Dr. Jatin Nagpal for comments on the manu-script, Dr. Malini Bajpai and Dr. Sreedhara Voleti forhelpful discussion during the course of this project.We thank Dr. Sunil Khattar for providing the GST-pFastBacHTb vector, Ms. Saima Aslam, and SanatanUpmanyu for technical assistance, Pankaj Gulati andAayush Seth for assistance with the FPLC purifica-tions.This research work was supported by RanbaxyLaboratories Limited, Gurgaon, Haryana.

The authors have declared no conflict of interest.

5 References

[1] Yanagi, S., Inatome, R., Takano, T., Yamamura, H., Syk ex-pression and novel function in a wide variety of tissues.Biochem. Biophys. Res. Commun. 2001, 288, 495–498.

[2] Chu, D. H., Morita, C. T., Weiss, A., The Syk family of proteintyrosine kinases in T-cell activation and development. Im-munol. Rev. 1998, 165, 167–180.

[3] Zhu, D. M., Tibbles, H. E., Vassilev, A. O., Uckun, F. M., SYKand LYN mediate B-cell receptor-independent calcium-in-duced apoptosis in DT-40 lymphoma B-cells. Leuk Lym-phoma. 2002, 43, 2165–2170.

[4] Zhang, J., Billingsley, M. L., Kincaid, R. L., Siraganian, R. P.,Phosphorylation of Syk activation loop tyrosines is essen-tial for Syk function. An in vivo study using a specific anti-Syk activation loop phosphotyrosine antibody. J. Biol. Chem.2000, 275, 35442–35447.

[5] Stenton, G. R., Kim, M. K., Nohara, O., Chen, C. F. et al.,Aerosolized Syk antisense suppresses Syk expression, me-diator release from macrophages, and pulmonary inflam-mation. J. Immunol. 2000, 164, 3790–3797.

[6] Matsuda, M., Park, J. G., Wang, D. C., Hunter, S. et al., Abro-gation of the Fc gamma receptor IIA-mediated phagocyticsignal by stem-loop Syk antisense oligonucleotides. Mol.Biol. Cell. 1996, 7, 1095–1106.

[7] Hueber, A. J., McInnes, I. B., Is spleen tyrosine kinase inhi-bition an effective therapy for patients with RA? Nat. Clin.Pract. Rheumatol. 2009, 5, 130–131.

[8] Taniguchi, T., Kobayashi, T., Kondo, J., Takahashi, K. et al.,Molecular cloning of a porcine gene syk that encodes a 72-kDa protein-tyrosine kinase showing high susceptibility toproteolysis. J. Biol. Chem. 1991, 266, 15790–15796.

[9] Papp, E., Tse, J. K., Ho, H., Wang, S. et al., Steady state kinet-ics of spleen tyrosine kinase investigated by a real time flu-orescence assay. Biochemistry 2007, 46, 15103–15114.

[10] Wong, B. R., Grossbard, E. B., Payan, D. G., Masuda, E. S.,Tar-geting Syk as a treatment for allergic and autoimmune dis-orders. Expert. Opin. Investig. Drugs. 2004, 13, 743–762.

[11] Yang, C.,Yanagi, S.,Wang, X., Sakai, K. et al., Purification andcharacterization of a protein-tyrosine kinase p72syk fromporcine spleen. Eur. J. Biochem. 1994, 221, 973–978.

[12] Zioncheck, T. F., Harrison, M. L., Isaacson, C. C., Geahlen, R.L., Generation of an active protein-tyrosine kinase fromlymphocytes by proteolysis. J. Biol. Chem. 1988, 263,19195–19202.

[13] Sahdev, S., Khattar, S. K., Saini, K. S., Production of active eu-karyotic proteins through bacterial expression systems: Areview of the existing biotechnology strategies. Mol. Cell.Biochem. 2008, 307, 249–264.

[14] Goldberg, J. M., Manning, G., Liu, A., Fey, P. et al., The dic-tyostelium kinome—analysis of the protein kinases from asimple model organism. PLoS Genet. 2006, 2, e38.

[15] Kikkawa, U., Mann, S. K., Firtel, R. A., Hunter, T., Molecularcloning of casein kinase II alpha subunit from Dictyosteliumdiscoideum and its expression in the life cycle. Mol. Cell. Biol.1992, 12, 5711–5723.

[16] Moreno-Bueno, G., Cales, C., Behrens, M. M., Fernandez-Re-nart, M., Isolation and characterization of casein kinase Ifrom Dictyostelium discoideum. Biochem. J. 2000, 349,527–537.

[17] Tan, J. L., Spudich, J.A., Dictyostelium myosin light chain ki-nase. Purification and characterization. J. Biol. Chem. 1990,265, 13818–13824.

[18] Arya, R., Bhattacharya, A., Saini, K. S., Dictyostelium dis-coideum—A promising expression system for the produc-tion of eukaryotic proteins. FASEB J. 2008, 22, 4055–4066.

[19] Arias-Palomo, E., Recuero-Checa, M.A., Bustelo, X. R., Llor-ca, O., 3D structure of Syk kinase determined by single-par-ticle electron microscopy. Biochim. Biophys. Acta 2007, 1774,1493–1499.

[20] Sada, K.,Takano,T.,Yanagi, S.,Yamamura, H., Structure andfunction of Syk protein-tyrosine kinase. J. Biochem. 2001,130, 177–186.

[21] Siraganian, R. P., Zhang, J., Suzuki, K., Sada, K., Protein ty-rosine kinase Syk in mast cell signaling. Mol. Immunol. 2002,38, 1229–1233.

[22] Kurosaki, T., Johnson, S. A., Pao, L., Sada, K. et al., CambierJC. Role of the Syk autophosphorylation site and SH2 do-mains in B cell antigen receptor signaling. J. Exp. Med. 1995,182, 1815–1823.

[23] Tsang, E., Giannetti, A. M., Shaw, D., Dinh, M. et al., Molecu-lar mechanism of the Syk activation switch. J. Biol. Chem.2008, 283, 32650–32659.

[24] Arya, R., Aslam, S., Gupta, S., Bora, R. S. et al., Productionand characterization of pharmacologically active recombi-nant human phosphodiesterase 4B in Dictyostelium dis-coideum. Biotechnol. J. 2008, 3, 938–947.

[25] Berman, H. M., Bhat, T. N., Bourne, P. E., Feng, Z. et al., TheProtein Data Bank and the challenge of structural ge-nomics. Nat. Struct. Biol. 2000, 7, 957–959.

[26] Jain,A. N., Surflex: Fully automatic flexible molecular dock-ing using a molecular similarity-based search engine. J.Med. Chem. 2003, 46, 499–511.

[27] Yamamoto, N., Hasegawa, H., Seki, H., Ziegelbauer, K. et al.,Development of a high-throughput fluoroimmunoassay forSyk kinase and Syk kinase inhibitors. Anal. Biochem. 2003,315, 256–261.

[28] Furlong, M. T., Mahrenholz, A. M., Kim, K. H., Ashendel, C.L. et al., Identification of the major sites of autophosphory-lation of the murine protein-tyrosine kinase Syk. Biochim.Biophys. Acta 1997, 1355, 177–190.

[29] Baldock, D., Graham, B., Akhlaq, M., Graff, P. et al., Purifica-tion and characterization of human Syk produced using abaculovirus expression system. Protein. Expr. Purif. 2000,18, 86–94.

[30] Lupher, M. L., Jr., Rao, N., Lill, N. L., Andoniou, C. E. et al.,Cbl-mediated negative regulation of the Syk tyrosine ki-nase. A critical role for Cbl phosphotyrosine-binding do-

Biotechnol. J. 2010, 5 www.biotechnology-journal.com

BiotechnologyJournal Biotechnol. J. 2010, 5

12 © 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

main binding to Syk phosphotyrosine 323. J. Biol. Chem.1998, 273, 35273–35281.

[31] Arias-Palomo, E., Recuero-Checa, M.A., Bustelo, X. R., Llor-ca, O., Conformational rearrangements upon Syk auto-phosphorylation. Biochim. Biophys. Acta 2009, 1794, 1211–1217.

[32] Catalina, M. I., Fischer, M. J., Dekker, F. J., Liskamp, R. M. etal., Binding of a diphosphorylated-ITAM peptide to spleentyrosine kinase (Syk) induces distal conformationalchanges: A hydrogen exchange mass spectrometry study. J.Am. Soc. Mass. Spectrom. 2005, 16, 1039–1051.

[33] Geahlen, R. L., Syk and pTyr’d: Signaling through the B cellantigen receptor. Biochim. Biophys. Acta 2009, 1793, 1115–1127.

[34] Law, C. L., Sidorenko, S. P., Chandran, K. A., Draves, K. E. etal., Molecular cloning of human Syk. A B cell protein-tyro-sine kinase associated with the surface immunoglobulin M-B cell receptor complex. J. Biol. Chem. 1994, 269,12310–12319.

[35] Manning, G., Plowman, G. D., Hunter, T., Sudarsanam, S.,Evolution of protein kinase signaling from yeast to man.Trends Biochem. Sci. 2002, 27, 514–520.

[36] Atwell, S., Adams, J. M., Badger, J., Buchanan, M. D. et al., Anovel mode of Gleevec binding is revealed by the structureof spleen tyrosine kinase. J. Biol. Chem. 2004, 279,55827–55832.