A Premature Termination of Human Epidermal Growth Factor ... · TheScientificWorldJournal 3...

Research ArticleA Premature Termination of Human Epidermal Growth FactorReceptor Transcription in Escherichia coli

Jihene Elloumi-Mseddi1 Karim Jellali1 Antonio Villalobo2 and Sami Aifa1

1 Centre of Biotechnology of Sfax PO Box 1177 3018 Sfax Tunisia2 Instituto de Investigaciones Biomedicas Consejo Superior de Investigaciones Cientıfica and Universidad Autonoma de MadridArturo Duperier 4 28029 Madrid Spain

Correspondence should be addressed to Sami Aifa samiaifacbsrnrttn

Received 4 April 2014 Revised 25 July 2014 Accepted 28 July 2014 Published 15 October 2014

Academic Editor Adhar C Manna

Copyright copy 2014 Jihene Elloumi-Mseddi et al This is an open access article distributed under the Creative Commons AttributionLicense which permits unrestricted use distribution and reproduction in any medium provided the original work is properlycited

Our success in producing an active epidermal growth factor receptor (EGFR) tyrosine kinase in Escherichia coli encouraged usto express the full-length receptor in the same host Despite its large size we were successful at producing the full-length EGFRprotein fused to glutathione S-transferase (GST) that was detected by Western blot analysis Moreover we obtained a majoritariantruncated GST-EGFR form detectable by gel electrophoresis and Western blot This truncated protein was purified and confirmedby MALDI-TOFTOF analysis to belong to the N-terminal extracellular region of the EGFR fused to GST Northern blot analysisshowed two transcripts suggesting the occurrence of a transcriptional arrest

1 Introduction

Since its discovery the epidermal growth factor receptor(EGFR) continues to be the subject of myriad investigationsin cancer signalling EGFR is a transmembrane proteincomposed of an extracellular region containing the ligand-binding site a transmembrane 120572-helix and an intracellularregion harboring the tyrosine kinase domain that is flankedby a juxtamembrane domain (JM) and a C-terminal tailThe majority of solid tumor types overexpress at least oneof the EGFR family members (HER1EGFR HER2NeuHER3 and HER4) [1] As a consequence the EGFR fam-ily has been targeted by many antibodies for therapeuticpurpose as many of them were approved to treat differentcancer types (ie breast colorectal and non-small cellslung carcinoma (NSCLC) among others) [2] Moreovermany tyrosine kinase inhibitors (TKI) competing with ATPbinding were used to antagonize these receptors and someof them were approved also for treating some cancer typessuch as NSCLC [3] Progress has been done in understand-ing the EGFR activation process especially with researchaided by the crystallization of distinct segments of thereceptor

The ligand-induced dimerization of the EGFR wasrevealed by independent crystal structure studies show-ing that both juxtaposed monomers are maintaining aldquobutterflyrdquo-like dimeric structure via a protruding loop fromeach domain II [4 5]

Stamos et al solved the EGFR tyrosine kinase crys-tal structure encouraging the exploration of the relation-ship between the active ectodomain (ECD) conformationalchange and the intracellular activation of the tyrosine kinase(TK) domain [6] Many studies have tried to understandthe dimerization of the intracellular region of the EGFRleading to autophosphorylation showing the importance ofthe calmodulin-binding site located at the juxtamembrane(JM) segment in this process [7ndash9] An allosteric mechanismof EGFR TK activation was proposed based on the formationof an asymmetric dimer of the EGFR kinase domain [10]where the JM region was shown to be the activator of theallosteric mechanism [11ndash13]

The ECD is subdivided into four domains domains I andIII are indispensable for ligand binding and domains II andIV play a crucial role in receptor dimerization and activation

There are 12 potential N-linked glycosylation sites inthe receptor two in each of domains I and II and four in

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 830923 6 pageshttpdxdoiorg1011552014830923

2 The Scientific World Journal

each of domains III and IV [14] It was shown that theglycosylation process is related to ligand-binding acquisition[15] and core glycosylation plays an important role in bothEGF binding and kinase activation [16] Treatment with tuni-camycin yielded a 130 kDa aglycosylated form of the EGFRwith altered ligand-binding capacity poor kinase activationand aberrant subcellular localization of the receptor [17]Interestingly the combination of tunicamycin and erlotinibcausedmore inhibitory effect on EGFR phosphorylation thanthat induced by erlotinib alone [18] Tsuda et al suggestedthat the sugar chain linked to Asn-420 of the ECD plays acrucial role in EGF binding and prevents the spontaneousoligomerization of the EGFR [19] which otherwise maylead to uncontrollable receptor activation Moreover it wasshown that core glycosylation is responsible for EGFRvIIIself-dimerization [20]

Heterologous expression of the intracellular EGFRdomain [21 22] or the ECD of some EGFR family memberswas also attained in Escherichia coli and thereafter purifiedyielding an active recombinant tyrosine kinase [22] thatallowed the preparation of antibodies [23 24]

In the present work we have modified the E coliculture conditions in order to improve the quantity andquality of recombinant full-length and a truncated segmentof the EGFR expressed as a fusion with the GST tag Therecombinant proteins could be used for the production ofcorresponding anti-EGFR antibodies

2 Materials and Methods

21 Strains and Reagents E coli strain BL21 CodonPlus(Stratagene) was used for GST-fusion protein expression andJM109 competent bacteria (Promega) were used for plasmidconstruction and maintenance The vector pLXSN contain-ing the full-length human EGFR was a gift from ProfessorAxel Ullrich (Max Planck Institute Martinsried Germany)E coli expression vector pGEX-6P-1 was purchased fromAmersham Pharmacia Biotech Anti-EGFR (sc-03) and anti-GST (sc-459) antibodies were obtained from Santa-CruzBiotechnology The horseradish peroxidase conjugated anti-rabbit and anti-mouse IgG antibodies were purchased fromPromega

22 Plasmid Construction The DNA fragment coding forthe full-length EGFR was amplified by PCR using Pfu-polymerase (Stratagene) and the pLXSN-EGFR plasmid astemplate The following oligonucleotides were used for PCRamplification 51015840-GA GTC GAC CGA TGC GAC CCT CCGGGA C-31015840 as forward primer with a SalI site underlinedand 51015840-GAGCG GCC GCCCTCCGTGGT TCATGCTCCA-31015840 as a reverse primer with a NotI site underlined Theobtained fragment was double digested by SalI-NotI andinserted in pGEX-6P-1 We used S-300 columns (AmershamPharmacia Biotech) to purify PCR products and a Qiagenkit (QIAquick PCR purification kit) to remove restrictionenzymes fromdigestedDNAbefore ligation using the ldquoready-to-gordquo T4 DNA ligase (Amersham Pharmacia Biotech) Theresulting construct named pGEX-EGFR was analyzed by

restriction enzymes and DNA sequencing to ascertain itscorrectness

23 Recombinant Protein Expression Analysis and Dimer-ization E coli BL21 strains were grown overnight inLuriarsquos broth or M9 minimal medium containing ampicillin(75 120583gmL) IPTG induction expression analysis and GSTproteins immobilization on glutathione-Sepharose 4B beadswere performed as previously described [22]

The dimerisation assaywas performed on 20120583g of proteinextract incubatedwith 150120583MBS3 for 10min at 4∘Cor 025Mglutaraldehyde for 1min at room temperature The reactionwas stopped respectively by the SDS-PAGE loading bufferor 06M glycine

The proteins were then analysed on SDS-PAGE followedby a Western blot using anti-EGFR or anti-GST antibodies

24 In-Gel Protein Digestion and Sample Preparation forMALDI-TOFTOF Analysis The bands of interest fromCoomassie colloidal-stained 1D gel were excised manuallydeposited in 96-well plates and processed automaticallyin a Proteineer DP (BrukerDaltonics Bremen Germany)The digestion protocol used was based on Shevchenko etal [25] with minor variations gel plugs were washed firstwith 50mM ammonium bicarbonate then with acetonitrile(ACN) prior to reduction with 10mM dithiothreitol in25mM ammonium bicarbonate solution Alkylation wascarried out afterwards with 55mM 3-120573-indole acrylic acid(IAA) in 50mM ammonium bicarbonate Gel pieces wererinsed with 50mM ammonium bicarbonate and thereafterwith ACN and were dried under a stream of nitrogenModified porcine trypsin (sequencing grade PromegaMadi-son WI) was added at a final concentration of 16 ng120583Lin 25 ACN50mM ammonium bicarbonate solution andthe digestion was incubated at 37∘C for 6 h The reactionwas stopped by adding 05 trifluoroacetic acid (TFA) Thetryptic-eluted peptideswere dried by speed-vacuumcentrifu-gation and were resuspended in 4 120583L of MALDI solution A08 120583L aliquot of each peptide mixture was deposited onto a389-well OptiTOF Plate (Applied Biosystems FraminghamMA) and allowed to dry at room temperature Matrix solu-tion (3mgmL 120572-cyano-4-hydroxycinnamic acid (CHCA) inMALDI solution) was then added (08120583L) and the mixturewas dried at room temperature

25 MALDI Peptide Mass Fingerprinting MSMS Analysisand Database Searching For MALDI-TOFTOF analysissamples were automatically acquired in an ABi 4800 MALDITOFTOFmass spectrometer (Applied Biosystems Framing-hamMA) in positive ion reflectormode (the ion accelerationvoltage was 25 kV for MS acquisition and 1 kV for MSMS)and the obtained spectra were stored into the ABi 4000Series Explorer Spot Set Manager Peptide mass finger-printing (PMF) and mass spectrometrymass spectrometry(MSMS) fragment ion spectra were smoothed and correctedto zero baseline using routines embedded in ABi 4000 SeriesExplorer Software v36 Each PMF spectrum was internallycalibrated with the mass signals of trypsin autolysis ions

The Scientific World Journal 3

asymp50kDa

GST

(kDa)

97

66

45

30

1 2 3 4 5 6 M

Figure 1 GST-EGFR expression in E coli BL21 strain The proteinswere separated in a 10 SDS-PAGE and stained with Coomassieblue (Lanes 1 and 2) Two clones of E coli BL21 cells transformed byempty pGEX-6P-1 vector (Lanes 3ndash6)Different clones of E coliBL21cells transformed by pGEX-EGFR (Lane M) The protein masses ofthe BioRad markers are indicated in kDa The arrows point to GSTand the asymp50 kDa protein

to reach a typical mass measurement accuracy of lt25 ppmKnown trypsin and keratin mass signals as well as potentialsodium and potassium adducts (+21Da and +39Da resp)were removed from the peak list To submit the combinedPMF and MSMS data to MASCOT software v21 (MatrixScience LondonUK)GPSExplorer v49was used searchingin the nonredundant NCBI protein database

26 E coli Total RNA Extraction and Northern Blot Anal-ysis After solubilization cells were solubilized in Eurosol(EuroClone) solution and chloroform was added in theproportion of 1 10 After mixing and 5-minute incubationon wet ice the sample was centrifuged for 15min at 12000 gThe upper aqueous phase containing RNAwas then collectedand precipitated with cold isopropanol for 15min on wet iceAfter centrifugation at 12000 g at 4∘C the total RNA pelletwas washed with 75 ethanol and centrifuged at 8000 g for15min at 4∘C Northern blot analysis was performed accord-ing to Sambrook and Russel [26] A PCR fragment corre-sponding to the EGFR ECD was [32P]dCTP-radiolabelled byRediprime II Random Prime Labelling System (Amersham)and used as a probe to detect EGFR messengers

3 Results

31 Expression of Recombinant EGFR in FusionwithGST TheDNA fragment encoding the open reading frame (ORF) ofthe EGFRwas amplified using pLXSN-EGFR as template andinserted in frame with the ORF of the GST in pGEX-6P-1The obtained construct pGEX-EGFR was used to transformBL21 CodonPlus In order to optimize the expression offull-length GST-EGFR we have varied the culture mediaand analyzed the produced proteins in each condition byWestern blot After cultureinduction of the recombinantstrains as described in Section 2 cells were solubilized inloading buffer and total proteinswere analyzed in SDS-PAGEThe analysis displays amajor asymp50 kDa band corresponding toa truncated form of the EGFR (Figure 1)The detection of thistruncated formwas confirmed byWestern blot analysis using

the anti-GST as primary antibody (Figure 2(a)) Moreovera longer exposition to ECL allowed the detection of a160 kDa band corresponding to the full-length GST-EGFR(Figure 2(b)) The full-length GST-EGFR was also detectedby the anti-EGFR antibody especially after immobilization inglutathione beads of the recombinant protein derived from Ecoli grown in M9 minimal medium (Figure 2(c))

The dimerization of the receptor was also studied viaBS3 chemical cross-linking and glutaraldehyde Our resultsshowed that the full-length GST-EGFR preserved the abilityof forming dimers even in the absence of EGF (data notshown)

32 Analysis of the GST-EGFR Truncated Form In orderto understand the origin of EGFR truncation and checkthe integrity of its ORF the asymp50 kDa truncated GST-EGFR protein overproduced in all media used was puri-fied from the SDS-PAGE as described in Section 2 Aftertrypsin digestion this protein was subjected to MALDI-TOFTOF analysis (Figure 3) Many peaks from the obtainedspectrum were identified as peptides belonging to theGST or the EGFR proteins using the Mascot search tool(httpwwwmatrixsciencecom) (Figure 3) The identifica-tion of the obtained peptides showed that the truncated pro-tein starts from the GST and stops at domain II of the EGFRaccording to the matched peptides (Figure 3(c)) Accordingto the last matched peptide the truncation corresponds tothe first 293 amino acids of EGFR protein sequence whichbecomes 530 residues with the GSTThe size of this truncatedprotein (asymp50 kDa) was determined by the Expazy proteomictools (PeptideMass at httpwwwexpasyorgtools)The cor-responding mRNA size was 1590 bp (879 bp corresponds tothe EGFR RNA)

Northern blot analysis using a PCR probe correspondingto the EGFRECD showed the presence of two transcriptsThesmall one is present even in noninduced cells but the large oneis produced after IPTG stimulation only (Figure 4)

These results suggest that the largest transcript encodesthe full-length GST-EGFR while the smaller one expressedeven in the absence of IPTG induction is probably encodingthe truncated protein The presence of the small transcriptunder noninduced condition could be explained by theldquoleakyrdquo expression encountered with the pGEX vectors asdescribed by the supplier

4 Discussion

The production of EGFR protein in an easy and less costinghost could facilitate enormously the production of specificantibodies or screening for tyrosine kinase antagonists Forlong time E coli has been an ideal heterologous expressionhost characterized by the multiplicity of developed strainsand the continuous productionpurification improvement ofthe recombinant proteinsWehave previously succeededwiththe expression andproduction of an active intracellular EGFRdomain [22] which encouraged our present work We nowachieved expressing the full-length EGFR as a GST-fusionprotein that was detectable only by Western blot analysis


1 2 3 4

(a)

2 3

(b)

asymp50kDa

160kDa

GST 275kDa

P E

(c)

Figure 2 Western blot analysis of GST-EGFR Samples from E coli expressing GST (lane 1) and GST-EGFR (lanes 2ndash4) were processed byWestern blot with the anti-GST antibody (a) 5min ECL exposure (b) 30min ECL exposure (c) the protein extract was immobilized onglutathione-Sepharose beads run on SDS-PAGE and analyzed by Western blot using the anti-EGFR antibody (Lane P) Protein extract of Ecoli cells transformed with empty pGEX-6P-1 (Lane E) Protein extract of E coli cells transformed with pGEX-EGFR after immobilization onglutathione beads Arrows indicate the position of each expressed recombinant protein

asymp50kDa

(kDa)

97664530

201144

(a)

10090807060

4050

30201007960 1317

Mass (mz)

4 18388

109456

190492

99E+

203473

119255

23602 28816 34030

Inte

nsity

()

(b)

2 MRPSGTAGAA LLALLAALCP ASRALEEKKV CQGTSNKLTQ LGTFEDHFLS

151 SNNPALCNVE SIQWRDIVSS DFLSNMSMDF QNHLGSCQKC DPSCPNGSCW

LQRMFNNCEV VLGNLEITYV QRNYDLSFLK TIQEVAGYVL IALNTVERIP51101 LENLQIIRGN MYYENSYALA VLSNYDANKT GLKELPMRNL QEILHGAVRF

GAGEENCQKL TKIICAQQCS GRCRGKSPSD CCHNQCAAGC TGPRESDCLV201 CRKFRDEATC KDTCPPLMLY NPTTYQMDVN PEGKYSFGAT CVKKCPRNYV251 VTDHGSCVRA CGADSYEMEE DGVRKCKKCE GPCRKVCNGI GIGEFKDSLS 301 INATNIKHFK NCTSISGDLH ILPVAFRGDS FTHTPPLDPQ ELDILKTVKE 351 ITGFL401

(c)

Figure 3 Identification of the truncated protein by MALDI-TOFTOF analysis (a) The gel in the inset shows the purified N-terminaltruncated EGFR with its corresponding size The protein masses of the BioRad markers are indicated in kDa (b) The spectrum showsdifferent peaks of theMS analysis done byMALDI-TOFTheN-terminal truncated EGFR (asymp50 kDa) was subjected to in-gel trypsin digestionbefore MALDI-TOF analysis The arrows show the peaks that served for MSMS analysis with their corresponding masses used for proteinidentification (c) MASCOT results matched peptides to EGFR sequence are shown in capital bold the last identified peptide ends at K293

Moreover chemical cross-linkingwith BS3 showed that EGFRdimerization is preserved in E coli

The expression of EGFR was concomitant with the pro-duction of the asymp50 kDa truncated protein that was analyzedin MALDI-TOFTOF and the MASCOT search confirmedits identity corresponding to a truncation within the ECDdomain II of the receptor The origin of this truncation was

analyzed by Northern blot and the presence of two tran-scripts was detected a larger one that was dependent on IPTGstimulation of the promoter and is more probably encodingthe full-length GST-EGFR protein and a shorter transcriptthat is present even in the absence of IPTG induction andcould encode the truncated protein The presence of thissmall transcript in noninduced cells is very likely due to


1 2 3 4

(a)

1 2 3 4

(b)

Figure 4 Northern blot analysis of EGFR expression (a) Total RNA(20 120583g) was loaded on a formaldehyde 15 agarose gel RNA wasextracted from E coliBL21 cells transformedwith pGEX-EGFR afterone-hour induction with 01mM IPTG (lane 1) or without induction(lane 2) Extracted RNA of E coli BL21 cells transformed with emptypGEX-6P-1 after one-hour induction with 01mM IPTG (lane 3) orwithout IPTG induction (lane 4) used as control (b)The gel inAwasblotted on a nitrocellulose membrane and radioactive hybridizationwas performed with a 19 kb PCR fragment of EGFR ECD cDNA

a premature transcriptional termination signal within theEGFR cDNA although this arrest was not total since thedetection of the full-length transcript and protein was appar-entThis could be explained by the existence of a transcriptionattenuation phenomenon due to E coli-like leaky terminatorsignals within the EGFR cDNA sequence We have scannedthe EGFR cDNA sequence by several online programs andfound two overlapping hairpin structures resembling those ofthe tryptophan operon In fact the sequence of EGFR cDNA(starting from the ATG) 1220ndashttcaggct-1227 hybridizes withthe sequence 1331-aagtccga-1324 in a first palindrome andwith the sequence 1236-aagtccg-1230 in another palindromeFavoring one of these two palindromes could be responsiblefor the present transcription attenuation

Furtherwork should be performed to establish themolec-ular basis of this premature termination in transcription ofthe full-length EGFR and on the possibility of bypassingthis arrest in order to express larger quantities of the full-length receptor Meanwhile the overexpression of the 50 kDatruncated protein could serve itself for the production ofanti-EGFR antibodies directed against the N-terminal of thereceptor

5 Conclusions

The adoption of E coli as a host of heterologous expressionis still a primordial strategy for any gene functional orimmunogenicity study The present work demonstrates thatthis strategy is valuable also for transmembrane proteins likereceptor tyrosine kinases which could facilitate proteomic

studies Moreover encountering an E coli-like transcriptionarrest signal within a human oncogene could have manyimpacts Since EGFR has its viral homolog ErbB one canspeculate the occurrence of DNA transfer from prokaryoticto eukaryotic genomes through virusesWe have to search foranalogous results of expression to check our hypothesis

Conflict of Interests

The authors declare that there is no conflict of interests

Acknowledgments

Theauthors thank Professor Axel Ullrich for the EGFR cDNAgiftThey also thank Dr Vivian de Los Rıos from the Serviciode Espectrometrıa de Masas of the Centro de InvestigacionesBiologicas (CSIC) for the MALDI-TOFTOF experimentsThisworkwas supported by theMinistry ofHigher Educationand Scientific Research of Tunisia and the SpanishAgency forInternational Cooperation and Development (AECID)

References

[1] OHamid ldquoEmerging treatments in oncology focus on tyrosinekinase (erbB) receptor inhibitorsrdquo Journal of the AmericanPharmacists Association vol 44 no 1 pp 52ndash58 2004

[2] J Elloumi K Jellali I Jemel and S Aifa ldquoMonoclonal antibod-ies as cancer therapeuticsrdquo Recent Patents on Biotechnology vol6 no 1 pp 45ndash56 2012

[3] I Jemel K Jellali J Elloumi and S Aifa ldquoThe offer of chemistryto targeted therapy in cancerrdquo Recent Patents on Biotechnologyvol 5 no 3 pp 174ndash182 2011

[4] H Ogiso R Ishitani O Nureki et al ldquoCrystal structure ofthe complex of human epidermal growth factor and receptorextracellular domainsrdquo Cell vol 110 no 6 pp 775ndash787 2002

[5] T P J Garrett N M McKern M Lou et al ldquoCrystal structureof a truncated epidermal growth factor receptor extracellulardomain bound to transforming growth factor 120572rdquo Cell vol 110no 6 pp 763ndash773 2002

[6] J Stamos M X Sliwkowski and C Eigenbrot ldquoStructure of theepidermal growth factor receptor kinase domain alone and incomplex with a 4-anilinoquinazoline inhibitorrdquo The Journal ofBiological Chemistry vol 277 no 48 pp 46265ndash46272 2002

[7] J Martın-Nieto and A Villalobo ldquoThe human epidermalgrowth factor receptor contains a juxtamembrane calmodulin-binding siterdquo Biochemistry vol 37 no 1 pp 227ndash236 1998

[8] S Aifa J Aydin G Nordvall I Lundstrom S P S SvenssonandO Hermanson ldquoA basic peptide within the juxtamembraneregion is required for EGF receptor dimerizationrdquo ExperimentalCell Research vol 302 no 1 pp 108ndash114 2005

[9] S Aifa N Miled F Frikha M R Aniba S P S SvenssonandA Rebai ldquoElectrostatic interactions of peptides flanking thetyrosine kinase domain in the epidermal growth factor receptorprovides a model for intracellular dimerization and autophos-phorylationrdquo Proteins Structure Function and Genetics vol 62no 4 pp 1036ndash1043 2006

[10] X Zhang J Gureasko K Shen P A Cole and J Kuriyan ldquoAnallosteric mechanism for activation of the kinase domain ofepidermal growth factor receptorrdquo Cell vol 125 no 6 pp 1137ndash1149 2006


[11] K W Thiel and G Carpenter ldquoEpidermal growth factor recep-tor juxtamembrane region regulates allosteric tyrosine kinaseactivationrdquo Proceedings of the National Academy of Sciences ofthe United States of America vol 104 no 49 pp 19238ndash192432007

[12] N Jura N F Endres K Engel et al ldquoMechanism for activationof the EGF receptor catalytic domain by the juxtamembranesegmentrdquo Cell vol 137 no 7 pp 1293ndash1307 2009

[13] M Red Brewer S H Choi D Alvarado et al ldquoThe juxtamem-brane region of the EGF receptor functions as an activationdomainrdquoMolecular Cell vol 34 no 6 pp 641ndash651 2009

[14] S Bishayee ldquoRole of conformational alteration in the epidermalgrowth factor receptor (EGFR) functionrdquo Biochemical Pharma-cology vol 60 no 8 pp 1217ndash1223 2000

[15] L J Slieker T M Martensen and M D Lane ldquoSynthesisof epidermal growth factor receptor in human A431 cellsglycosylation-dependent acquisition of ligand binding activityoccurs post-translationally in the endoplasmic reticulumrdquo TheJournal of Biological Chemistry vol 261 no 32 pp 15233ndash152411986

[16] A Ullrich L Coussens J S Hayflick et al ldquoHuman epidermalgrowth factor receptor cDNA sequence and aberrant expressionof the amplified gene in A431 epidermoid carcinoma cellsrdquoNature vol 309 no 5967 pp 418ndash425 1984

[17] A M Soderquist and G Carpenter ldquoGlycosylation of the epi-dermal growth factor receptor in A-431 cells the contributionof carbohydrate to receptor functionrdquo The Journal of BiologicalChemistry vol 259 no 20 pp 12586ndash12594 1984

[18] Y H Ling T Li R Perez-Soler and M Haigentz Jr ldquoActi-vation of ER stress and inhibition of EGFR N-glycosylationby tunicamycin enhances susceptibility of human non-smallcell lung cancer cells to erlotinibrdquo Cancer Chemotherapy andPharmacology vol 64 no 3 pp 539ndash548 2009

[19] T Tsuda Y Ikeda andNTaniguchi ldquoTheAsn-420-linked sugarchain in human epidermal growth factor receptor suppressesligand-independent spontaneous oligomerization possible roleof a specific sugar chain in controllable receptor activationrdquoJournal of Biological Chemistry vol 275 no 29 pp 21988ndash219942000

[20] H Fernandes S Cohen and S Bishayee ldquoGlycosylation-induced conformational modification positively regulatesreceptor-receptor association a study with an aberrantepidermal growth factor receptor (EGFRvIIIΔEGFR)expressed in cancer cellsrdquo Journal of Biological Chemistryvol 276 no 7 pp 5375ndash5383 2001

[21] J G Koland K Maddison OrsquoBrien and R A Cerione ldquoExpres-sion of epidermal growth factor receptor sequences as E colifusion proteins applications in the study of tyrosine kinasefunctionrdquo Biochemical and Biophysical Research Communica-tions vol 166 no 1 pp 90ndash100 1990

[22] J Elloumi-Mseddi K Jellali and S Aifa ldquoIn vitro activation andinhibition of recombinant EGFR tyrosine kinase expressed inEscherichia colirdquo The Scientific World Journal vol 2013 ArticleID 807284 5 pages 2013

[23] Y Lin Z Liu J Jiang Z Jiang Y Ji and B Sun ldquoExpression ofintracellular domain of epidermal growth factor receptor andgeneration of its monoclonal antibodyrdquo Cellular amp MolecularImmunology vol 1 no 2 pp 137ndash141 2004

[24] X Liu Z He M Zhou et al ldquoPurification and characterizationof recombinant extracellular domain of human HER2 fromEscherichia colirdquo Protein Expression and Purification vol 53 no2 pp 247ndash254 2007

[25] A Shevchenko M Wilm O Vorm and M Mann ldquoMassspectrometric sequencing of proteins from silver-stained poly-acrylamide gelsrdquo Analytical Chemistry vol 68 no 5 pp 850ndash858 1996

[26] J Sambrook and D W RusselMolecular Cloning A LaboratoryManual Cold Spring Harbor Laboratory Press Cold SpringHarbor NY USA 3rd edition 2001

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each of domains III and IV [14] It was shown that theglycosylation process is related to ligand-binding acquisition[15] and core glycosylation plays an important role in bothEGF binding and kinase activation [16] Treatment with tuni-camycin yielded a 130 kDa aglycosylated form of the EGFRwith altered ligand-binding capacity poor kinase activationand aberrant subcellular localization of the receptor [17]Interestingly the combination of tunicamycin and erlotinibcausedmore inhibitory effect on EGFR phosphorylation thanthat induced by erlotinib alone [18] Tsuda et al suggestedthat the sugar chain linked to Asn-420 of the ECD plays acrucial role in EGF binding and prevents the spontaneousoligomerization of the EGFR [19] which otherwise maylead to uncontrollable receptor activation Moreover it wasshown that core glycosylation is responsible for EGFRvIIIself-dimerization [20]

Heterologous expression of the intracellular EGFRdomain [21 22] or the ECD of some EGFR family memberswas also attained in Escherichia coli and thereafter purifiedyielding an active recombinant tyrosine kinase [22] thatallowed the preparation of antibodies [23 24]

In the present work we have modified the E coliculture conditions in order to improve the quantity andquality of recombinant full-length and a truncated segmentof the EGFR expressed as a fusion with the GST tag Therecombinant proteins could be used for the production ofcorresponding anti-EGFR antibodies

2 Materials and Methods

21 Strains and Reagents E coli strain BL21 CodonPlus(Stratagene) was used for GST-fusion protein expression andJM109 competent bacteria (Promega) were used for plasmidconstruction and maintenance The vector pLXSN contain-ing the full-length human EGFR was a gift from ProfessorAxel Ullrich (Max Planck Institute Martinsried Germany)E coli expression vector pGEX-6P-1 was purchased fromAmersham Pharmacia Biotech Anti-EGFR (sc-03) and anti-GST (sc-459) antibodies were obtained from Santa-CruzBiotechnology The horseradish peroxidase conjugated anti-rabbit and anti-mouse IgG antibodies were purchased fromPromega

22 Plasmid Construction The DNA fragment coding forthe full-length EGFR was amplified by PCR using Pfu-polymerase (Stratagene) and the pLXSN-EGFR plasmid astemplate The following oligonucleotides were used for PCRamplification 51015840-GA GTC GAC CGA TGC GAC CCT CCGGGA C-31015840 as forward primer with a SalI site underlinedand 51015840-GAGCG GCC GCCCTCCGTGGT TCATGCTCCA-31015840 as a reverse primer with a NotI site underlined Theobtained fragment was double digested by SalI-NotI andinserted in pGEX-6P-1 We used S-300 columns (AmershamPharmacia Biotech) to purify PCR products and a Qiagenkit (QIAquick PCR purification kit) to remove restrictionenzymes fromdigestedDNAbefore ligation using the ldquoready-to-gordquo T4 DNA ligase (Amersham Pharmacia Biotech) Theresulting construct named pGEX-EGFR was analyzed by

restriction enzymes and DNA sequencing to ascertain itscorrectness

23 Recombinant Protein Expression Analysis and Dimer-ization E coli BL21 strains were grown overnight inLuriarsquos broth or M9 minimal medium containing ampicillin(75 120583gmL) IPTG induction expression analysis and GSTproteins immobilization on glutathione-Sepharose 4B beadswere performed as previously described [22]

The dimerisation assaywas performed on 20120583g of proteinextract incubatedwith 150120583MBS3 for 10min at 4∘Cor 025Mglutaraldehyde for 1min at room temperature The reactionwas stopped respectively by the SDS-PAGE loading bufferor 06M glycine

The proteins were then analysed on SDS-PAGE followedby a Western blot using anti-EGFR or anti-GST antibodies

24 In-Gel Protein Digestion and Sample Preparation forMALDI-TOFTOF Analysis The bands of interest fromCoomassie colloidal-stained 1D gel were excised manuallydeposited in 96-well plates and processed automaticallyin a Proteineer DP (BrukerDaltonics Bremen Germany)The digestion protocol used was based on Shevchenko etal [25] with minor variations gel plugs were washed firstwith 50mM ammonium bicarbonate then with acetonitrile(ACN) prior to reduction with 10mM dithiothreitol in25mM ammonium bicarbonate solution Alkylation wascarried out afterwards with 55mM 3-120573-indole acrylic acid(IAA) in 50mM ammonium bicarbonate Gel pieces wererinsed with 50mM ammonium bicarbonate and thereafterwith ACN and were dried under a stream of nitrogenModified porcine trypsin (sequencing grade PromegaMadi-son WI) was added at a final concentration of 16 ng120583Lin 25 ACN50mM ammonium bicarbonate solution andthe digestion was incubated at 37∘C for 6 h The reactionwas stopped by adding 05 trifluoroacetic acid (TFA) Thetryptic-eluted peptideswere dried by speed-vacuumcentrifu-gation and were resuspended in 4 120583L of MALDI solution A08 120583L aliquot of each peptide mixture was deposited onto a389-well OptiTOF Plate (Applied Biosystems FraminghamMA) and allowed to dry at room temperature Matrix solu-tion (3mgmL 120572-cyano-4-hydroxycinnamic acid (CHCA) inMALDI solution) was then added (08120583L) and the mixturewas dried at room temperature

25 MALDI Peptide Mass Fingerprinting MSMS Analysisand Database Searching For MALDI-TOFTOF analysissamples were automatically acquired in an ABi 4800 MALDITOFTOFmass spectrometer (Applied Biosystems Framing-hamMA) in positive ion reflectormode (the ion accelerationvoltage was 25 kV for MS acquisition and 1 kV for MSMS)and the obtained spectra were stored into the ABi 4000Series Explorer Spot Set Manager Peptide mass finger-printing (PMF) and mass spectrometrymass spectrometry(MSMS) fragment ion spectra were smoothed and correctedto zero baseline using routines embedded in ABi 4000 SeriesExplorer Software v36 Each PMF spectrum was internallycalibrated with the mass signals of trypsin autolysis ions


asymp50kDa

GST

(kDa)

97

66

45

30

1 2 3 4 5 6 M




3 Results







4 Discussion



1 2 3 4

(a)

2 3

(b)

asymp50kDa

160kDa

GST 275kDa

P E

(c)


asymp50kDa

(kDa)

97664530

201144

(a)

10090807060

4050

30201007960 1317

Mass (mz)

4 18388

109456

190492

99E+

203473

119255

23602 28816 34030

Inte

nsity

()

(b)





(c)






1 2 3 4

(a)

1 2 3 4

(b)




5 Conclusions





Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


asymp50kDa

GST

(kDa)

97

66

45

30

1 2 3 4 5 6 M




3 Results







4 Discussion



1 2 3 4

(a)

2 3

(b)

asymp50kDa

160kDa

GST 275kDa

P E

(c)


asymp50kDa

(kDa)

97664530

201144

(a)

10090807060

4050

30201007960 1317

Mass (mz)

4 18388

109456

190492

99E+

203473

119255

23602 28816 34030

Inte

nsity

()

(b)





(c)






1 2 3 4

(a)

1 2 3 4

(b)




5 Conclusions





Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4

(a)

2 3

(b)

asymp50kDa

160kDa

GST 275kDa

P E

(c)


asymp50kDa

(kDa)

97664530

201144

(a)

10090807060

4050

30201007960 1317

Mass (mz)

4 18388

109456

190492

99E+

203473

119255

23602 28816 34030

Inte

nsity

()

(b)





(c)






1 2 3 4

(a)

1 2 3 4

(b)




5 Conclusions





Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology


1 2 3 4

(a)

1 2 3 4

(b)




5 Conclusions





Acknowledgments


References



































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

























Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

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