Journal of Immunological Methods xxx (2014) xxx–xxx
JIM-11923; No of Pages 11
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Auto-induction for high yield expression of recombinant novelisoallergen tropomyosin from King prawn (Melicertuslatisulcatus) for improved diagnostics and immunotherapeutics
Martina Koeberl a,b,c,e, Sandip D. Kamath a,b,c,e, Shruti R. Saptarshi a,b,c,e, Michael J. Smout b,d,e,Jennifer M. Rolland f,g, Robyn E. O'Hehir f,g, Andreas L. Lopata a,b,c,e,⁎a Molecular Immunology Group, James Cook University, Townsville, QLD, Australiab Centre for Biodiscovery and Molecular Discovery of Therapeutics, James Cook University, Townsville, QLD, Australiac Comparative Genomic Centre, James Cook University, Townsville, QLD, Australiad Queensland Tropical Health Alliance, James Cook University, Cairns, QLD, Australiae Australian Institute of Tropical Health and Medicine, James Cook University, Townsville, QLD, Australiaf Department of Immunology, Monash University, Melbourne, Victoria, Australiag Department of Allergy, Immunology and Respiratory Medicine, The Alfred Hospital and Monash University, Melbourne, Victoria, Australia
Please cite this article as: Koeberl, M., etropomyosin from King prawn (Melicertus
a b s t r a c t
Article history:Received 15 August 2014Received in revised form 20 October 2014Accepted 20 October 2014Available online xxxx
Food allergies are increasing worldwide, demonstrating a considerable public health concern.Shellfish allergy is one of the major food groups causing allergic sensitization among adults andchildren, affecting up to 2% of the general world population. Tropomyosin (TM) is the majorallergen in shellfish and frequently used in the diagnosis of allergic sensitization and the detectionof cross-contaminated food. To improve and establish better and more sensitive diagnostics forallergies and immunotherapeutics, large quantities of pure allergens are required. To establish areproducible method for the generation of pure recombinant tropomyosin we utilized in thisstudydifferent Escherichia coli strains (NM522, TOP10 andBL21(DE3)RIPL). In addition, isopropyl-β-D-thiogalactoside (IPTG) induction was compared with a novel auto-induction system to allowthe generation of larger quantities of recombinant allergen.We demonstrated that the B-strain ofE. coli is better for the expression of TM compared to the K-strain. Moreover, a higher yield couldbe achieved when using the auto-induction system, with up to 62 mg/l. High yield expressedrecombinant TM fromKing prawn (KP)was compared to recombinant TM from Black tiger prawn(Penm 1). We demonstrated that recombinant TM from KP and known isoallergen Penm 1 havevery similar molecular and immunological characteristics. Overall, we demonstrate that auto-induction can be used to express larger quantities of recombinant allergens for the developmentof diagnostic, to quantify allergens as well as immunotherapeutics employing isoallergens.
Keywords:Auto-inductionTropomyosinRecombinant allergenKing prawnAllergy diagnosticsEscherichia coli strains (K and B)
yl-β-D-thiogalactoside;ant tropomyosin; KP,monodon) Black tigeride gel electrophoresis.g 21, Room001, James.: +61 7 4781 4563;
ata).
t al., Auto-induction folatis..., J. Immunol. Meth
1. Introduction
Food allergies are increasing worldwide and thereforerepresenting a growing public health concern. Consequently,the diagnosis of food allergy and the detection of allergens infood are of increasing importance. Subsequently, the standard-ization of allergen preparations is of great importance.Recombinant allergenic proteins therefore become an essentialtool for the detection of allergens (Abdel Rahman et al., 2010;
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Kamath et al., 2014b; Koeberl et al., 2014), the diagnosis ofallergic sensitization (Bohle andVieths, 2004; Jeong et al., 2009;Lopata and Kamath, 2012; Shreffler, 2011; van der Ventel et al.,2011; Vrtala et al., 2014) and immunotherapeutic applications(Burks et al., 2013;Nowak-Węgrzyn and Sampson, 2011; Vrtalaet al., 2014). Moreover, the production of recombinantisoallergens allows species-specific investigation of allergens.Although similar speciesmight have a similar genome, differentallergens (isoallergens) can be expressed as demonstrated forfish, house dust mite and shellfish (Abramovitch et al., 2013;Sharp et al., 2014; Vrtala et al., 2014). Isoallergens can causedifferent IgE binding of allergic patients. Therefore the investi-gation and expression of isoallergens are necessary for betterdiagnostic and the development of immunotherapeutics.
Shellfish allergy is one of the major food allergies, includingvarious species of the crustacean and mollusks. Allergicreaction towards shellfish can range from mild reactions tolife threatening anaphylaxis, affecting 2% of the world popula-tion (Lopata et al., 2010). Tropomyosin (TM) is the majorallergen in shellfish and is an isoallergen as well as a pan-allergen that is cross-reactive with mites, cockroaches andnematodes (Lopata and Lehrer, 2009; Lopata et al., 2010).Therefore TM is frequently used in the diagnosis of allergicsensitization and detection of allergens in cross-contaminatedfood. To investigate and understand the molecular andimmunological cross-reactivity of TM better, recombinanttropomyosins from different species have been reported inthe literature. Allergic tropomyosins investigated belong to fivedifferent arthropod groups. These groups include (1) prawns(Bauermeister et al., 2011; Kamath et al., 2013, 2014a,b; Leunget al., 1994, 1996), (2) crabs (Abramovitch et al., 2013; Leunget al., 1998; Liang et al., 2008), (3) house dust mites (Aki et al.,1994, 1995; Asturias et al., 1998; Pittner et al., 2004),(4) cockroaches (Jeong et al., 2004; Santos et al., 1999;Satinover et al., 2005) and (5) nematodes (Alvite and Esteves,2009; Asturias et al., 2000; Hartmann et al., 2006; Sereda et al.,2010). In most studies TM was expressed in Escherichia coli(E. coli) with the exception of German cockroach TM (Jeonget al., 2004) and chicken TM (Hilario et al., 2001), beingexpressed in yeast.
E. coli is a widely used expression system for the productionof recombinant proteins, thus it has a well-characterizedgenome, established techniques for genetic manipulation andit is easy and inexpensive to cultivate (Graeslund et al., 2008a,b; Papaneophytou and Kontopidis, 2014; Papaneophytou et al.,2013; Xu et al., 2012). Historically, E. coli are divided into twomajor strains, the K-strain and the B-strain (Daegelen et al.,2009). Both strains are commonly used in research andindustry (Shibahara et al., 2010), while both strains seem tohave advantages and disadvantages. Both strains grow similar-ly when cultured, however, the production of by-products(such as acetic acid, formic acid, and lactic acid) seems to differ(Waegeman and De Mey, 2012; Yoon et al., 2012) and inaddition the B-strains seem to grow faster in comparison to K-strains. This can lead to a higher biomass and therefore higherprotein production (Waegeman and De Mey, 2012; Yoon et al.,2012). Consequently this would mean less unwanted by-products and possible inhibition of growth and expression ofheterologous proteins. The disadvantages of B-strains, howev-er, is that they can lose the plasmids (Waegeman and De Mey,2012).
Please cite this article as: Koeberl, M., et al., Auto-induction fotropomyosin from King prawn (Melicertus latis..., J. Immunol. Meth
E. coli are mainly cultured in Luria-Bertani (LB) media at37 °C and expression is chemically induced using isopropyl-β-D-thiogalactoside (IPTG). In 2005, a different concept, the so-called auto-induction, was introduced by Studier (Studier,2005). Auto-induction uses a media in which glucose, glyceroland lactose are present at the same time. E.coli prefers glucoseover lactose for growth, however, the consumption of glyceroland lactose follows when glucose is depleted. The auto-induction system is activated through the suppression of thelac operon by glucose. In turn, when glucose is metabolized,E. coli will utilize lactose and the lac operon will be activated.Therefore, if glucose and lactose are present in the mediasimultaneously, the induction will be delayed (Studier, 2005).
The efficiency of expressing recombinant TM (rTM) isonly reported in a few publications. rTM yields achieved arepublished for house dust mites (Aki et al., 1994; Asturias et al.,1998), cockroach (Jeong et al., 2004) and chicken (Hilario et al.,2001). Despite the different sources of TM and expressionsystems, the purified rTMyields reported are relatively low andvary between 7.2 mg/l (Jeong et al., 2004) and 26 mg/l(Asturias et al., 1998). However, larger quantities are neededas standards for the detection of allergens, diagnostics forallergic sensitization and development of immunotherapeutics.Studier (Studier, 2005) reported that auto-induction canincrease the protein yield by 10-fold over the IPTG induction,with up to 50 mg/l. To achieve this higher protein yield forallergens the current study compares for the first time thisnovel auto-induction systemwith conventional IPTG inductionto generate larger quantities of themajor shellfish allergen, TM.We report in this study the novel amino acid sequence of TMfromKing prawn (Melicertus latisulcatus). This novel TMof Kingprawn was used to compare the expression in E. coli K-strains(NM522, TOP10) and B-strain (BL21(DE3)RIPL) aswell as auto-induction versus IPTG induction. We established for the firsttime a high cell density culture using ZY-5052 auto-inducingmedium. Moreover, the molecular and immunological proper-ties of rTM from King prawn was compared to an isoallergen,the rTM of Black tiger prawn (Pen m 1).
2. Materials and methods
2.1. cDNA sequencing of King prawn TM
To investigate the amino acid sequence from TM of Kingprawn (M. latisulcatus (KP)) cDNAwas amplified and analyzed.The procedure of sequencing TM from KP was followed aspreviously published for Black tiger prawn (Kamath et al., 2013,2014a). Briefly, green headless KPwere obtained from the localmarket (Townsville, Australia) and transported to the labora-tory on ice. The total RNA was isolated from KP meat usingTRIzol® reagent (Life Technologies, Australia), with 0.1 g ofmuscle tissue in 1ml of reagent. cDNAwas generated fromRNAusing cDNA Synthesis kit (Bioline, Australia). The generatedcDNA was used as a template to amplify the coding region ofTM using forward (5′-GCGGATCCGACGCCATCAAGAAGAAGATGC-3′) and reverse (5′-GCGAATTCTTAGTAGCCAGACAGTTCGCTG-3′) primers. The PCR was run for 35 cycles, denaturation at95 °C for 0.45min, annealing 55 °C for 0.45min and elongationat 72 °C for 1 min. The generated PCR products were purifiedon a low melting agarose gel. The amplified PCR product wascloned into the sequencing vector pCR2.1 using the TOPO®
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Cloning Reaction (Invitrogen, USA) protocol and transformedinto chemical competent E. coli cells. Positive colonies werescreened by colony PCR using the gene specific oligonucleotideprimers for the presence of inserts. The plasmid DNA waspurified from overnight cultures using the QIAprep SPINreaction KIT (Qiagen, Germany) and products analyzed togenerate the nucleotide sequence of TM from King prawn byMacrogen sequence analysis.
The investigated cDNA sequence was converted into aminoacid sequence and published as Genbank accession numberJX171685. To examine differences in amino acid sequence of TMfrom KP, the amino acid sequence of TM from KP was alignedwith TM from Black tiger prawn (Penaeus monodon), Genbankaccession number HM486525, European house dust mite(Dermatophagoides pteronyssinus), Genbank accession numberACI32128 and American house dust mite (D. pteronyssinus),Genbank accession number Q23939.
2.2. Expression of rTM of King prawn
To express rTM the coding region for TM was cloned intoexpression vector pProEX HTb (kindly provided by JamesBurnell), using the restriction enzymes, BamH1 and EcoR1(Promega, Madison, Wisconsin, USA). Ligation of the codingregion into the expression vector was conducted using T4 DNALigase (Invitrogen, Carlsbad, CA, USA). The cloned vector wastransformed into chemically competent E. coli cells; (1) NM522(kindly provided by James Burnell), (2) BL21(DE3)RIPL (kindlyprovided by Patrick Schaeffer) or (3) TOP10 (Invitrogen, USA),using heat shock and incubation in SOC medium at 37 °C for1 h. The cells were grown overnight on LB agar with 100 μg/mlof ampicillin at 37 °C. The colonies were tested for the presenceof the insert by PCR. Plasmid and insert were confirmed byMacrogen sequence analysis, using QIAprep SPIN reaction KIT(Qiagen, Germany). Cells were stored in glycerol stock at−80 °C.
2.2.1. Isopropyl β-D-1-thiogalactopyranoside (IPTG) inductionTo investigate and optimize the expression of rTM from KP
in LBmedia with IPTG induction E. coli strainswere grown overvarious hours (0–24 h) and soluble and insoluble fractions ofbacteria culture analyzed. In detail, E. coli cells were grownovernight in LB media with 100 μg/ml of ampicillin at 37 °Cwhile shaking at 0.09 g. An aliquot of the overnight culture wastransferred into LB broth containing 100 μg/ml ampicillin andgrown until the culture reached middle to late log phase. Theexpression of rTM was inducted by adding 1 mM IPTG,subsequent 1 ml aliquots of cultures were analyzed over 24 h.These 1 ml aliquots were tested for the solubility of rTMaccording to Studier (Studier, 2005). Briefly, for testing rTM insoluble fraction the 1 ml aliquots were centrifuged (5 min,20,000 g) and supernatant analyzed by SDS-PAGE (10 μlloaded). The pellet of the insoluble fraction was re-suspendedin 5× SDS-PAGE loading buffer (30 μl) and separated by SDS-PAGE (20 μl loaded). As results of the tested 1 ml aliquots, theoptimizedmethodwas that the cellswere harvested after 5 h ofIPTG induction. The cells were harvested by centrifugation(20 min 17,000 g) and re-suspended in 10 ml extraction buffer(25mMTris–HCl, 300mMNaCl, pH 8) prior to applying Frenchpressure cell for cell lysis (Kamath et al., 2014b). Cell lysatewasagain centrifuged (20 min 25,000 g) and supernatant collected
Please cite this article as: Koeberl, M., et al., Auto-induction fotropomyosin from King prawn (Melicertus latis..., J. Immunol. Meth
(referred to as first supernatant). Cell lysate was re-suspendedin extraction buffer (5 ml) and centrifuged, thereby generatingthe second supernatant.
2.2.2. Auto-inductionThe method used for the auto-induction is based on a
previous study by Studier (Studier, 2005). In detail, the E. colistrains were grown in ZYP-0.8G medium with 100 μg/ml ofampicillin at 37 °C for 4 h. An aliquot was transferred into ZY-5052 media, containing 100 μg/ml ampicillin. Similar to theoptimization of IPTG induction, different time points (0–24 h)of growing bacteria culture were analyzed using 1 ml aliquots.The 1ml aliquotswere tested for soluble and insoluble fractionsas described above. Three different approaches were investi-gated to optimize the expression of rTM in auto-inductionmedia; (1) antifoamwas added to themedia, (2)metal contentwas diminished, (3) glucose concentration was increased by10-fold, with increase expression time of 48 h. In the optimizedmethod, the culture was grown overnight at 37 °C, for 18 hwith 0.13 g rotation and the media was composed of ZY-5052broth containing 100 μg/ml ampicillin, 100 μM MgSO4 (Sigma,USA), 100 μM Fe(III)Cl3 (Sigma, USA) and antifoam (10 μ/l)(Sigma, USA).
Auto-induction experiments were performed in two differ-ent volumes (250 ml and 1000 ml). For the expression in250 ml volume, the harvested and lysated cells were re-suspended in 10 ml extraction buffer (25 mM Tris–HCl,300 mM NaCl, pH 8) prior to applying French pressure cell.Subsequently the cell lysatewas centrifuged (20min 25,000 g),supernatant collected and cell lysate re-suspended again in theextraction buffer (5 ml) and centrifuged. This generates thesecond supernatant. The pellet was re-suspended in 10 mlextraction buffer, leading to the third supernatant. For theexpression in 1000 ml the volume of the extraction buffer wasincreased to 30mlprior to applying French pressure cell. All thesubsequent supernatants collected (12 supernatants in total)were re-suspended in 30 ml extraction buffer.
2.2.3. Purification of rTM of King prawnTo purify the expressed rTM from the supernatants affinity
chromatography was performed, using as a nickel-chargedmetal chelate column (GE Healthcare, USA). The His-Trappurification was performed on a Biologic Duoflow FPLC(BioRad, USA) using 250 mM imidazole as elution buffer. Thefractions collected from the FPLC containing the purifiedtropomyosin rTM were dialyzed in phosphate buffered saline(PBS) as previously performed by Kamath et al. (Kamath et al.,2014b). The concentration of recombinant protein generatedwas quantified using the Bradfords assay using bovine serumalbumin (BSA) as standard.
2.3. SDS-PAGE
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was performed to separate proteins on a 12%polyacrylamide gel. The protein components were resolved at200Vuntil the tracker dye reached the base using a BioRadMini-Protean Tetra cell (BioRad, USA). The gel was stained usingCoomassie brilliant blue R-250 to visualize the separatedproteins (Kamath et al., 2014a). To estimate the expressionlevel of rTM in cell lysate, densitometric analysis was performed
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using the Quantity One 1-D analytical software (BioRad)(Kamath et al., 2014a).
2.4. Immunoblotting
2.4.1. Immunoblotting with antibodiesTo confirm the presence of TM and rTM various antibodies
were used. After the proteins have been separated by SDS-PAGE, the proteins were transferred to an activated PVDFmembrane (BioRad, USA) and blocked with 5% skim milkpowder in PBSwith 0.05% Tween-20. For TMand rTMdetectionin various samples, the proteins were exposed to (1) monoclo-nal anti-tropomyosin antibody (Abcam, Cambridge, MA, USA)diluted 1:6000 and subsequently rabbit anti-mouse IgGhorseradish peroxidase labeled antibody (Sigma, USA) diluted1:40,000; (2) In-house (Kamath et al., 2014b) polyclonal anti-crustacean antibody diluted 1:30,000 and subsequently anti-rabbit IgG HRP conjugate (Promega, USA) diluted 1:40,000;(3) Anti-His antibody (GE Healthcare, UK) diluted 1:3000 andsubsequently rabbit anti-mouse IgG horseradish peroxidaselabeled antibody (Sigma, USA) diluted 1:40,000. Antibodybindingwas visualized using the enhanced chemiluminescence(ECL) kit (BioRad, USA).
2.4.2. Patient sera IgE immunoblottingIgE immunoblotting was performed to analyze patient IgE
antibody reactivity to rTM. Six subjects with a confirmedclinical history of allergic reactivity to shellfish and onenonatopic subject were recruited by The Alfred Hospital,Allergy Clinic, Melbourne Victoria, Australia (Johnston et al.,2014; Kamath et al., 2014a). 25 μg of the rTM were loaded on12% SDS-PAGE and resolved at 170 V until the tracker dyereached the bottom. The proteins were transferred to anactivated PVDF membrane and blocked with 5% skim milkpowder in PBS with 0.05% Tween-20 and subsequentlyincubated overnight with 1:10 diluted patient sera using aslot blot apparatus (Idea Scientific, USA). IgE binding wasdetected using rabbit anti-human IgE polyclonal antibody(diluted 1:10,000; (DAKO Corporation, USA)) and subsequent-ly goat-anti-rabbit IgG HRP conjugate (Promega, USA)(1:10,000) (Kamath et al., 2013, 2014a).
Ethics approval for this study was granted by James CookUniversity's Ethics committee (Project number H4313) incollaboration with The Alfred Hospital (Project number 192/07) and Monash University's Ethics Committees (MUHRECCF08/0225) (Kamath et al., 2014a).
2.5. Mass spectrometry
To confirm that the generated protein is TM, mass spectro-metric analysis was performed. A total of 200 μg of recombinantprotein was reduced with dithiothreitol at 60 °C for 30 min,alkylated in the dark with iodoacetamide at 37 °C for 30 min.This solution was loaded on a trypsin spin column (Sigma, USA),equilibrated and prepared according to the manufacturer'sinstructions. The loaded recombinant protein was incubated for15 min at room temperature and the digested peptides wereeluted twice with 100 μl 0.1% formic acid. The eluted peptideswere analyzed with an UPLC coupled with and ESI interface to aXevo TQMSmass spectrometer (Waters Corporation, Australia).The digested peptides were separatedwith a C18 1.7 μmcolumn
Please cite this article as: Koeberl, M., et al., Auto-induction fotropomyosin from King prawn (Melicertus latis..., J. Immunol. Meth
(Acquilty UPLC BEH300,Waters Corporation, Australia) at a flowrate of 0.4 ml/min and column temperature set at 45 °C. Thegradientwas run for 60min: 0–1min 2%B; 2.5–35min 10–50%B;35–47 min 50–90%B; 47–52 min 90%B; 52–60 min 2%B. Thecomposition of solvent A was 0.1% formic acid in water andsolvent B was 0.1% formic acid in acetonitrile. The time-of-flightanalyzer of the mass spectrometer was externally calibratedwith sodium formate from m/z 50 to 1990, with the data postacquisition lock mass-corrected using the monoisotopic mass ofthe lock spray (Leu-enkephalin). The uninterpreted data wereprocessed using ProteinLynx Global Server (PLGS) v2.3 (WatersCorporation, Australia) and converted into pkl files. Theconverted pkl files were searched with Mascot database usingvariable modifications of carbamidomethyl-C N-terminus,deamidation N, deamidation Q and oxidation of M. Up to fivemissed cleavage sites were allowed. Additional search parame-ters included a 0.1 Da tolerance against the database-generatedtheoretical peptide ion masses and a minimum of one matchedpeptide.
2.6. Protein extraction
Protein was extracted from King prawn and Black tigerprawn (P. monodon (BTP)). The outer shell of fresh prawnswasremoved and the abdominal muscles shredded into smallpieces and homogenized in 200 ml PBS. This slurry was mixedovernight at 4 °C followed by centrifugation at 3000 g for15min. The supernatant was filtered through a glass-fiber filterfollowed by 0.22 μm membrane filter (Millipore, Billerica, MA,USA) and stored at −80 °C until further use, named “raw-extract”. The second method, based on the natural exposure toprawn allergens included heating of thewhole raw prawnwithits outer shell in PBS at 100 °C for 15min, here named “whole-heated-extract”. “Whole-heated-extract” was then homoge-nized, mixed overnight (4 °C), centrifuged filtered and storedin the same way as “raw-extract”. BTP rTMwas expressed andpurified as described by Kamath et al. (Kamath et al., 2013,2014a,b)
2.7. CD spectrometry of recombinant tropomyosin
To compare the structures of generated rTM fromKP and rTMfrom BTP circular dichroism (CD) spectroscopy was performedas permethods previously described (Kamath et al., 2013, 2014a,b). Briefly, rTM samples were prepared in PBS, pH 7.2 andadjusted to a final concentration of 100 μg/ml. CD spectroscopywas performed on a J715Spectropolarimeter (Jasco, USA) withcontinuous nitrogen flushing at 25 °C. All measurements wereperformed using a 10 mm quartz-cuvette over a wavelengthrange of 190–260 nm. For wavelength analysis, the rTM sampleswere scanned with a step width of 0.2 nm and bandwidth of1 nm at 100 nm/min averaging over eight scans. Final data wereexpressed as mean residual ellipticity (θ) after subtracting thePBS blank spectrum (Kamath et al., 2014b).
3. Results and discussion
3.1. Comparison of TM amino acid sequences derived by cDNA
The amino acid sequence of TM from King prawn wasdeduced from the cDNA generated in this study and published
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as Genbank accession number JX171685. Moreover, TM fromKP was registered as allergen (Mel l 1) with the InternationalUnion of Immunological Societies.
TM is the major allergen in crustaceans, a well establishedpan-allergen and isoallergen. Fig. 1 displays the alignment ofamino acid sequences of TM from KP and is compared to TMfrom Black tiger prawn (BTP) and two isoallergens from twohouse dustmite species, Der p 10 and Der f 10. The amino acidshighlighted in red show the differences between KP and BTP,whereas the amino acids highlighted in yellow show thedifferences of house dust mite in comparison to KP. The aminoacid difference between the TM from the two very close relatedprawn species highlights the importance of isoallergens.Although species are genetically related, they can expressdifferent allergens. While the amino acid homology betweenKP and BTP is 95%, all the variances are between positions 34 to71. Moreover, as indicated by the black boxes (Fig. 1), aminoacids differ in one of the IgE antibody binding regions that wereidentified by Reese et al. (Reese et al., 2005) for Pen a 1. Thesefindings are important not only to understand immunologicalcross-reactivity between related species better, but in additionfor the development for more specific diagnostics and effectiveimmunotherapeutics.
3.2. Expression of rTM of King prawn
3.2.1. IPTG induced expression of rTMThe first aim was to optimize the expression time of rTM in
E. coli cells. NM522 and BL21(DE3)RIPL cells were grown in25 ml LB media and 1 ml aliquots were tested for theexpression of rTM. To visualize the expression 1 ml aliquotswere separated by SDS-PAGE, before and after IPTG induction.Moreover, to ensure the presence of soluble rTM, both solubleand insoluble fractions were tested. Supplementary Fig. 1visualizes the growing conditions of NM522 cells over a periodof 21 h. The BL21(DE3)RIPL performed very similar (data notshown). Three hours after IPTG was introduced, the concen-tration of rTM was high enough to be visualized by SDS-PAGE.In the following 2 h of growing the culture the amount of theexpressed rTM increased, however, the expression did notincrease considerably between 5 and 24 h. Interestingly, thedegradation of the soluble fraction after 21 h could be observedby immunoblotting analysis, using the anti-His antibody (datanot shown). Thereforewe focused on amore repeat productionof rTM after 5 h instead. Moreover, the presence of theexpressed protein was observed in the insoluble fraction, aspreviously reported by Asturias et al. (Asturias et al., 2000). No“leaky” expression was observed with either E. coli strain.
3.2.2. Auto-induction system for rTMThe first auto-induction experiments were similar to the
IPTG optimization. The changes made were that culturemedium had a higher OD600, in comparison to the IPTGinduced cells. Accordingly, 1 ml aliquots of cell cultures wereanalyzed for rTM in the soluble and insoluble fractions. Briefly,rTM was mainly present in the soluble fractions, however,some was formed in inclusion bodies. Although more proteinwas expressed, the amount of insoluble protein did notincrease in comparison to the IPTG induction (Table 1).
To further optimize the auto-induction system threedifferent approaches were investigated. (1) The addition of
Please cite this article as: Koeberl, M., et al., Auto-induction fotropomyosin from King prawn (Melicertus latis..., J. Immunol. Meth
antifoam increased the cell density and the cells could beharvested at higher OD600, leading to higher expression ofrTM. (2) The ZY-5052media contain differentmetals. However,chelate bindings between the His-tag of recombinant proteinand metals in the ZY-5052 media could be possible. Thereforean expression experiment without the addition of metal saltswas performed, however, adding MgSO4 and Fe(III)Cl3 seemsto be essential to generate expression of rTM. Moreover, nochelate binding with the addition of metals was observed bySDS-PAGE analysis before and after purification. (3) Glucoseconcentration was increased 10-fold, to investigate if celldensity and therefore expression can be further increased.Thus, glucose and lactose are present at the same time delayingthe induction (Sarduy et al., 2012; Studier, 2005). The increaseof glucose concentration in ZY-5052 media led to higher celldensity, but no increased rTM expression. The increase ofglucose concentration probably inhibited the lacoperon, even ifthe culture was grown for longer time (48 h).
NM522 cells demonstrated good expression when IPTGinduced, however, did not express rTM when being auto-induced. NM522 cells are Δ(lac-proAB) and therefore notsuitable for auto-induction. To be able to compare E. coli K-strain with E. coli B-strain TOP10 cells were used. Fig. 2 showsthat TOP10 cells can be used for auto-induction. However, incomparison to IPTG induced NM522 cells the expression is verylow, even after 24 h. The expression of rTM in TOP10 cells wasconfirmed by immunoblotting using anti-His antibody andpolyclonal anti-crustacean antibody. Densitometric analysisshowed that rTMcomprised only about 5.3% of the total proteinas demonstrated by SDS-PAGE. Fig. 2 demonstrates thatBL21(DE3)RIPL cells are expressing considerably more rTM.Therefore all further auto-induction experiments were har-vested after 18 h and grown under the conditions explained inthe material and method section.
3.2.3. IPTG induction versus auto-induction of rTM of King prawnTo compare the same experiments, the IPTG (NM522 and
BL21(DE3)RIPL) and auto-induction system (BL21(DE3)RIPL)using the same volume (250 ml) were performed. Table 1summarizes the most important findings for the differentinductions and bacteria strains investigated. The saturation inthe auto-inducing media was considerably higher than in theLB media under similar conditions, resulting in an increase inthe number of producing cells and therefore, in the finalconcentration of rTM (Sarduy et al., 2012; Sivashanmugamet al., 2009; Studier, 2005). The different fractions of thesupernatants were visualized by SDS-PAGE and the presence ofHis-TM confirmed using a specific anti-His antibody (Fig. 3AandB). In summary, the expression of rTMwas similar betweenthe NM522 and the BL21(DE3)RIPL cells using LB media. InFig. 3B one additional antibody binding band can be seen at75 kDa, representing the dimer of rTM.
To confirm that the expressed protein is TM, the purifiedrTM of each experiment was analyzed by mass spectrometry.Fig. 3C shows that for all experiments the expressed proteinwas identified as TM.Moreover, the sequence coverage of TM isexcellent and similar between the different experiments with71%, 70% and 77%, respectively. The peptides identified withmass spectrometry were distributed over the whole sequenceof TM. However, some peptides have been identified for one
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Fig. 1.Amino acid sequence alignment of tropomyosin fromKing prawn, Black tiger prawn, European house dustmite andAmerican house dustmite. The amino acids substitutions aremarked in red for differences between prawnsand yellow for differences between house dust mite and King prawn. Black boxes indicate IgE binding regions previously identified by Reese et al. (Reese et al., 2005) for the allergen Pen a 1. (For interpretation of the references tocolor in this figure legend, the reader is referred to the web version of this article.)
Table 1Summary of bacteria pellet and rTM yield generated for different E. coli strains and 250 ml LB (IPTG induction) or ZY-5052 auto-induction media investigated.
NM522(250 ml LB media)
BL21(DE3)RIPL(250 ml LB media)
BL21(DE3)RIPL(250 ml ZY-5052 media)
Optimal harvesting time 5 h after ITPG induction 5 h after ITPG induction 18 h after growing bacteria cultureOD 600 nm at optimal harvesting time 0.934 1.002 13.601g bacteria wet pellet 0.21 1.92 2.15% of rTM of total protein (Densitometric analysis based on Fig. 3A) 43% 54.46% 68.55%Ratio of insoluble rTM to total protein (Densitometric analysisbased on Fig. 3A)
10.23 8.06 7.7
Ratio of yield mg/g bacteria wet pellet 1.9 2.29 2.51Yield mg purified rTM 0.4 4.4 5.4
7M. Koeberl et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
but not the other TM, due to trypsin digestion and massspectrometry analysis.
3.2.4. Upscaling expression of by auto-inductionThe results in Table 1 show clearly that the auto-induction
using BL21(DE3)RIPL cells results in the highest rTM yield andwas used to upscale this system. The rTM expressed was in atotal volume of 1 l of ZY-5052 media using the optimized. Theseparation of different supernatants (12 supernatants in total)by SDS-PAGE can be seen in supplementary Fig. 2. Using 1 l ofZY-5052 media a total of 62 mg/l rTM could be purified. Thehigher yield achieved in the 1 l expression, compared to the250ml volume can be explained with higher cell density. Thus,the cell density is directly related to the culture conditions andthe medium. The yield achieved with the auto-inductionsystems for rTM is higher than that reported by Studier(Studier, 2005) with 30–50 mg/l. However, much lowerquantities were reported for cockroach with 7.2 mg/ml (Bal g7) (Jeong et al., 2004), 20 mg/ml for chicken (Hilario et al.,2001) expressed in yeast. Anisakis TMwith 15 mg/ml (Asturiaset al., 2000) and house dust mite (Der p 10) with 26 mg/ml
Fig. 2. SDS-PAGE protein analysis of bacterial cultures after NM522 IPTG induction, TOP(S) and insoluble (I) TM at different time points of culture growth. Lane numbers repre5052 18 h soluble; 5. TOP10 18 h insoluble; 6. TOP10 20 h soluble; 7. TOP10 20 h insolBL21(DE3)RIPL 18 h insoluble; 11. BL21(DE3)RIPL 20 h soluble; 12. BL21(DE3)RIPLinsoluble.
Please cite this article as: Koeberl, M., et al., Auto-induction fotropomyosin from King prawn (Melicertus latis..., J. Immunol. Meth
(Asturias et al., 1998) expressed in E. coli. With 62 mg/l ofprawn TM in our study the auto-induction systems generatedconsiderable more recombinant protein as previously reportedusing other systems and TMs.
3.3. Molecular and immunological comparison of rTM from Kingprawn and Black tiger prawn
To investigate the molecular and immunological propertiesof the generated rTM from KP, this protein was compared topreviously generated rTM form Black tiger prawn (Kamathet al., 2013, 2014a,b). Therefore protein from King prawnmuscle were extracted, as described in materials and methods.Moreover, to evaluate the allergenicity of TM of KP, it wascompared with isoallergen TM of Black tiger prawn (BTP).
TM of BTP is a registered allergen (Pen m 1) with theInternational Union of Immunological Societies and described asan allergen in various studies (Abramovitch et al., 2013; Kamathet al., 2013, 2014a,b). Fig. 4 shows the separated protein extractsfrom KP and BTP by SDS-PAGE and antibody binding using threedifferent antibodies; (1)monoclonal anti-tropomyosin antibody;
10 and BL21(DE3)RIPL cells by auto-induction. Fractionswere tested for solublesent: 1. Marker; 2. NM522 21 h soluble; 3. NM522 21 h insoluble; 4. TOP10 ZY-uble; 8. TOP10 24 h soluble; 9. BL21(DE3)RIPL ZY-5052 media 18 h soluble; 10.20 h insoluble; 13. BL21(DE3)RIPL 24 h soluble; and 14. BL21(DE3)RIPL 24 h
r high yield expression of recombinant novel isoallergenods (2014), http://dx.doi.org/10.1016/j.jim.2014.10.008
Fig. 3.Molecular and immunological comparison of different E. coli strains and induction systems. Proteins in generated fractions were separated by A) SDS-PAGE andconfirmation of expressed recombinant protein with B) anti-His tag antibody. The lane number represent analyzed supernatants of NM522 IPTG induction (Lane 2–4),BL21(DE3)RIPL cells for IPTG induction (Lane 5–7) and BL21(DE3)RIPL cells auto-induction (Lane 8–11). Lane numbers represent: 1. Marker; 2. 1st supernatant; 3. 2ndsupernatant; 4. pellet; 5. 1st supernatant; 6. 2nd supernatant; 7. pellet; 8. 1st supernatant; 9. 2nd supernatant; 10. 3rd supernatant; and 11. Pellet. The expressedproteins were further confirmed using mass spectrometry C). The summarized results of the mass spectrometry analysis, including sequence coverage, peptidesidentified and scores of purified TM of the different strain and induction system are displayed.
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(2) polyclonal anti-crustacean antibody; (3) anti-His antibody.In addition, the secondary structure of the recombinantallergen was compared to the rTM from BTP using CDspectrometry.
The SDS-PAGE profiles and antibody reactivities of the tworTM are very similar (Fig. 4A–D), with the exception of slightlystronger monoclonal anti-tropomyosin antibody to BTP. Asexpected, the anti-His antibody only bound to the rTM and notto the naturally occurring proteins (Fig. 4B). The rTM of KP andBTP share the same molecular weight by the SDS-PAGE(Fig. 4A), however, slightly higher than calculated from thesequence 36055.94 Da and for BTP 36978.91 Da. In Fig. 4E and Fthe CD spectrometric analysis of both rTM is compared anddemonstrate identical secondary structures. rTMs exhibited adistinct negative signal at 208 and 222 nm, typical for an alphahelical protein. The immunological reactivity of TM and rTMfromBTPhas previously been elucidated and is similar to nativeTM (Kamath et al., 2013, 2014a,b).
Fig. 5A and B illustrate the IgE antibody reactivity of sixpatients to rTM of KP and BTP. The allergic patients demon-strate very similar IgE binding, with the exception of patient 2,recognizing only rTM from BTP.
As presented in Fig. 1, the only region where the twoisoallergens differ are between amino acid positions 34 to 71. In
Please cite this article as: Koeberl, M., et al., Auto-induction fotropomyosin from King prawn (Melicertus latis..., J. Immunol. Meth
2005, Reese et al. (2005) identified one IgE binding epitopewithin this region at positions 42–55 for Pen a 1. In the currentstudy patient 2 is only recognizing the isoallergen of BTP andwe therefore expect very specific IgE binding to an epitope inthis region, possible the same as identified by Reese et al.(Reese et al., 2005). Species-specific reactivity to crustaceanshas previously been demonstrated for Black tiger prawncompared with Fresh water shrimp (Jirapongsananuruk et al.,2008) and White shrimp compared with Brown shrimp(Morgan et al., 1989), highlighting the need for generationrecombinant isoallergens for better management of patientswith specific-specific allergic sensitisation. Due to the compar-ison of KP extracts and rTM with the BTP analogs, we suggestthat TM is a major allergen in KP. While the molecular andimmunological properties of both isoallergens are very similarthe species-specific reactivity is of great importance forcomponent resolved diagnosis.
4. Conclusion
The current diagnostic tools for the detection of allergensin cross-contaminated food and the confirmation of allergicsensitization need to be improved. Therefore, recombinantallergenic proteins become an essential tool as well as for the
r high yield expression of recombinant novel isoallergenods (2014), http://dx.doi.org/10.1016/j.jim.2014.10.008
Fig. 4. SDS-PAGE and immunoblot analysis of protein extracts and r TM fromKingprawn and Black tiger prawn.Different protein extracts of KP andBTP are separated byA) SDS-PAGE and the presence of recombinant protein demonstrated by B) anti-His tag antibody C) monoclonal anti-tropomyosin antibody D) polyclonal rabbit anti-crustacean Lane numbers represent: 1. Marker; 2. rTM KP; 3. rTM BTP; 4. KP “whole-cooked-extract”; 5. BTP “whole-cooked-extract”; 6. KP “raw-extract”; and 7. BTP“raw-extract”. CD spectrometric analysis of rTM of KP (E) and rTM BTP (F).
9M. Koeberl et al. / Journal of Immunological Methods xxx (2014) xxx–xxx
component resolved diagnosis for immunotherapeutic ap-plications. Tropomyosin (TM), the major allergen in shell-fish, is a known pan-allergen and different isoallergens havebeen identified. In this study, the novel isoallergen of Kingprawn (KP) was sequenced and expressed for the first timeas recombinant protein. The yield of expressed functional
Fig. 5. Immunoblot analysis of serum IgE antibody from patients with allergy to crustaPatient 2; 4. Patient 3; 5. Patient 4; 6. Patient 5; 7. Patient 6; and 8. negative control se
Please cite this article as: Koeberl, M., et al., Auto-induction fotropomyosin from King prawn (Melicertus latis..., J. Immunol. Meth
recombinant allergen was increased using B-Strain of E. coliand a specific auto-induction system. This is the firstreported study to demonstrate that high yield recombinantallergen can be expressed using this auto-induction system.The molecular and immunological characteristics betweenthe TM isoallergen of KP and BTP are very similar, however,
cean. A) rTM KP B)rTM BTP, Lane numbers represent: 1. Marker; 2. Patient 1; 3.rum.
r high yield expression of recombinant novel isoallergenods (2014), http://dx.doi.org/10.1016/j.jim.2014.10.008
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species-specific IgE reactivity can be demonstrated. Thegeneration of good evaluated recombinant isoallergens isessential for the development of better diagnostics andimmunotherapeutics.
Acknowledgments
The National Measurement Institute (NMI) provided finan-cial support and scholarship for MK. AL is a holder of anAustralian Research Council (ARC) Future Fellowship.
Appendix A. Supplementary data
Supplementary data to this article can be found online athttp://dx.doi.org/10.1016/j.jim.2014.10.008.
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