Cloning, Auto-induction Expression, and Purification of rSpaA Swine Erysipelas Antigen\nDownload PDF\n369 Accesses\n8 Citations\nExplore all metrics\nAbstract\nThis work reports the cloning, expression, and purification of a 42-kDa fragment of the SpaA protein from Erysipelothrix rhusiopathiae, the main antigenic candidate for a subunit vaccine against swine erysipelas.\n\nThe use of an auto-induction protocol to improve heterologous protein expression in recombinant Escherichia coli cultures was also investigated.\n\nThe cellular growth pattern and metabolite formation were evaluated under different induction conditions.\n\nThe His-tagged protein was over-expressed as inclusion bodies, and was purified by a single chromatography step under denaturing conditions.\n\nAuto-induction conditions were shown to be an excellent process strategy, leading to a high level of rSpaA expression (about 25 % of total cellular protein content) in a short period of time.\n\nSimilar content being viewed by others\nRecombinant production of ESAT-6 antigen in thermoinducible Escherichia coli: the role of culture scale and temperature on metabolic response, expression of chaperones, and architecture of inclusion bodies\nArticle 04 June 2019\nA microbial expression system for high-level production of scFv HIV-neutralizing antibody fragments in Escherichia coli\nArticle Open access 22 October 2019\nHigh-yield production in Escherichia coli and convenient purification of a candidate vaccine against SARS-CoV-2\nArticle Open access 26 September 2022\nUse our pre-submission checklist\nAvoid common mistakes on your manuscript.\n\nIntroduction\nSwine erysipelas is a common disease in swine population around the world.\n\nIt causes arthritic lesions and endocarditic problems, which may result in carcasses being condemned, incurring substantial economic losses [1].\n\nThe disease is routinely controlled using cellular vaccines prepared with freeze-dried live cells of an attenuated strain of Erysipelothrix rhusiopathiae or using formalin-killed cells of virulent strains.\n\nHowever, it has been reported that exposure of animals to whole E. rhusiopathiae cells can aggravate arthritic lesions [2, 3].\n\nFurthermore, the available vaccines are able to prevent the acute, but not the chronic, form of erysipelas [3].\n\nThere is therefore an urgent need for the development of new, more efficient formulations that are free of cells.\n\nSubunit vaccines (made from one or more purified antigens) are good candidates for this purpose since they can trigger a protective immune response without any need to expose the immune system to the entire pathogenic agent.\n\nThere have been many efforts to identify E. rhusiopathiae antigens with several studies reporting that vaccination with the SpaA protein is able to induce protection in animals [4–6].\n\nThis indicates that SpaA could be a promising candidate for a subunit vaccine formulation against swine erysipelas.\n\nThe SpaA fragment has already been reported to protect pigs against challenge with serotypes 1 and 2, the most important serotypes in the swine industry [4].\n\nOther antigens tested as vaccine candidates in challenge experiments against virulent E. rhusiopathiae strains include recombinant fragments of RspA and RspB adhesion proteins, where RspA offered partial protection in murine model [7].\n\nOther surface proteins such as SpaB and SpaC were also tested with a recombinant N-terminal fragment of SpaC conferring protection in swine and mouse models [8].\n\nSubunit vaccines are usually manufactured using genetically modified organisms, which enable production of large amounts of recombinant antigen, while avoiding the cultivation of pathogenic organisms.\n\nEscherichia coli has been the host of choice for the majority of the recombinant proteins produced, and the lac promoter has been extensively used to control heterologous protein expression.\n\nA variety of different induction strategies have been proposed to improve protein production using this system.\n\nThe innovative auto-induction process proposed by Studier [9] can be used as a simple and reliable induction strategy for recombinant protein production, under the control of the lac promoter, in shake flask E. coli cultures.\n\nThe auto-induction medium is composed of a well-balanced mixture of different carbon sources and other important nutrients, allowing cells to grow to high densities, with expression of high levels of the heterologous protein.\n\nThis medium contains glucose, which encourages fast cellular growth in the early stages of the culture, while also inhibiting expression leakage by catabolic repression.\n\nLactose is also present, and is consumed after glucose depletion, activating the lac promoter to induce recombinant protein expression.\n\nGlycerol is included to (together with lactose) provide sustained growth during the induction phase.\n\nUsing this formulation, recombinant protein expression levels can be higher than those achieved using IPTG as inducer [9].\n\nIn this work, the DNA fragment that encodes an antigenic portion of the SpaA protein from E. rhusiopathiae was cloned in E. coli cells.\n\nIts expression was performed under auto-induction, followed by rSpaA purification to optimize the production of this candidate subunit vaccine against swine erysipelas.\n\nAntibodies produced against the recombinant protein were used to assess protein production during different induction or auto-induction procedures in shake flasks.\n\nMaterials and Methods\nMicroorganisms and Cloning Procedures\nCells of E. rhusiopathiae NCTC11002 were cultivated overnight at 37 °C in modified Feist medium [10], and 5 mL of the culture broth was used for genomic DNA extraction, as described previously [1].\n\nAmplification of the 1,026-bp fragment coding for the E. rhusiopathiae SpaA antigenic region [4] from the genomic DNA was performed using the sense primer SpaA_For–NcoI (5’ CATGCCATGG GC TACCAAAGTTTCGAAGC 3’) anchoring at nucleotide position (nt) 268 of the spaA gene, and anti-sense primer SpaA_Rev-HindIII (5’ CCCAAGCTT ATCTTTAGGTTTTTCTT 3’) anchoring at nt 1293, both designed based on DDBJ/EMBL/GenBank database sequences (accession number AB019124).\n\nThe underlined nucleotides indicate the NcoI and HindIII sites, respectively.\n\nThe nucleotides corresponding to the spaA sequence are shown in bold letters.\n\nThe addition of bases GC (italics) following the NcoI site at SpaA_For–NcoI primer was done to place the spaA gene sequence in frame with the ATG translation start codon present in NcoI restriction site.\n\nA map of the recombinant vector is shown in Fig.\n\n1.\n\nThe PCR amplification reaction was performed in a final volume of 50 μL using 0.5 μg of E. rhusiopathiae chromosomal DNA as the template, 0.2 mM of a dNTP mixture, 2.0 mM MgCl2, 0.2 μM of each primer, 1 unit of Taq DNA Polymerase (Fermentas), and appropriately diluted reaction buffer.\n\nThe conditions used for amplification were: initial denaturation step at 94 °C for 5 min, followed by 30 cycles of denaturation at 94 °C for 1 min, annealing at 55 °C for 1 min, extension at 72 °C for 3 min, and a final extension step at 72 °C for 10 min.\n\nThe amplified fragment was purified from the agarose gel, cloned into the cloning vector pGEM-T (Promega), and subsequently transformed in E. coli DH5α-competent cells.\n\nRecombinant cells were selected in agar plates containing 100 μg mL−1 ampicillin, and the transformation was confirmed by colony PCR using the primers described previously.\n\nThe insert had its sequence confirmed by the Sanger dideoxy method [11] and was transferred to the expression vector pET28a (Novagen) between NcoI and HindIII sites.\n\nUsing this system, a hexa-histidine tag was placed at the C-terminus of the recombinant protein.\n\nThe construct was used to transform E. coli BL21(DE3) cells by heat shock, and was further sequenced in both directions.\n\nAll procedures were performed according to the standard protocols [12].\n\nExpression of Recombinant SpaA\nGrowth, metabolite formation, and heterologous protein expression with E. coli BL21(DE3) bearing the construct pET28a_spaA were investigated in shake flask cultures employing different induction strategies.\n\nExperiments were performed using ZYM-5052 medium (with or without auto-induction) [9] or modified LB medium.\n\nThe modified LB medium was composed of the following components (in g L−1): 10.0 tryptone, 5.0 yeast extract, 5.0 NaCl, 10.0 glucose, 10.5 K2HPO4, and 3.7 MgSO4.\n\nThe ZYM-5052 medium was formulated with the following components (in g L−1): 0.5 glucose, 2.0 lactose, 5.0 glycerol, 10.0 tryptone, 5.0 yeast extract, 8.95 Na2HPO4, 3.4 KH2PO4, 0.5 MgSO4, 2.67 NH4Cl, 0.71 Na2SO4, and 1x metal solution.\n\nThe metal stock solution (1,000×) was composed of 50 mM FeCl3, 20 mM CaCl2, 10 mM each of MnCl2 and ZnSO4, and 2 mM each of CoCl2, CuCl2, NiCl2, Na2MoO4, Na2SeO3, and H3BO3, dissolved in HCl.\n\nKanamycin (30 μg mL−1) was added at 4-hour intervals during the cultivation.\n\nFour cultures were run simultaneously (experiments M1, M2, M3, and M4), with different induction protocols.\n\nExperiments were performed in duplicate.\n\nThe same inoculum was used for all four experiments, which was prepared from a single colony, freshly transformed, inoculated into 15 mL of LB medium, and incubated overnight at 37 °C and 250 rpm.\n\nExperiment M1 was performed using modified LB medium and IPTG induction.\n\nExperiment M2 corresponded to the auto-induction condition using complete ZYM-5052 medium.\n\nFor conditions M3 and M4, ZYM-5052 without lactose was used.\n\nIn experiment M3, protein expression was induced by a lactose pulse, and in experiment M4 an IPTG pulse was employed.\n\nIn previous tests using LB medium and IPTG induction at OD600nm of 0.6, there was cessation of growth before OD600nm of 2.5 was reached.\n\nSince cell growth can be greater in ZYM-5052 medium [9], induction was performed at OD600nm of 5.0 for conditions M1, M3, and M4.\n\nIPTG induction employed a final IPTG concentration of 0.25 mM.\n\nExperiments were performed using 1.0-L baffled flasks, after transferring 2 mL of inoculum to 100 mL of fresh medium with incubation at 37 °C and 250 rpm.\n\nCellular growth was monitored by measurements of the optical density (OD, λ = 600 nm) of the culture broth.\n\nMetabolite concentrations were measured using a Waters HPLC system fitted with an Aminex HPX-87H column (Bio-Rad) with 5 mM sulfuric acid as the mobile phase at a flow rate of 0.6 mL min−1.\n\nThe temperature was 50 °C.\n\nOrganic acids were detected at 210 nm (Waters 486 UV detector), while glucose and glycerol were measured with a refractive index detector (Waters 410).\n\nGlucose was also monitored throughout the cultivation using a glucose oxidase reaction kit (Laborlab, Brazil).\n\nPurification of Recombinant SpaA\nThe recombinant rSpaA was produced mainly in the form of inclusion bodies, which were solubilized under denaturing conditions before purification by affinity chromatography.\n\nThe purification protocol was initiated by washing 0.5 g of wet cells in 15 mL of lysis buffer containing 50 mM Tris–HCl (pH 7.4) and 25 % (w/v) sucrose and centrifuging at 5,000×g (at 4 °C).\n\nThe cells were then re-suspended again in 15 mL of lysis buffer, and left under gentle agitation for 30 min at 4 °C.\n\nLysozyme was added to a final concentration of 0.1 % (w/v), followed by incubation of the suspension at 4 °C for 35 min.\n\nThe cells were disrupted by sonication (VibraCell Sonics, 130 W, 20 kHz, 20 % amplitude, 8 pulses of 1 min at 30 s intervals).\n\nNucleic acids were degraded by adding 50 μg of DNAse and incubating for 30 min at 4 °C.\n\nInclusion bodies were then recovered by centrifuging at 18,000×g for 30 min at 4 °C.\n\nThe pellet was re-suspended in 15 mL of washing buffer containing 100 mM Tris–HCl (pH 8.0), 1.0 % (v/v) Triton X-100, and 3 M urea, and left under gentle agitation for 1 h at room temperature.\n\nAfter this period, the solution was again centrifuged (18,000×g for 30 min at 4 °C), and the washing procedure was repeated twice using the remaining pellet.\n\nFinally, the pelletized material was re-suspended in 4 mL of solubilization buffer (50 mM Tris–HCl at pH 7.5, 6 M guanidine hydrochloride, and 10 mM DTT), and left under gentle agitation for 16–20 h at 4 °C.\n\nThe methodology was based on previously reported procedures [12, 13], with modifications.\n\nPurification of the recombinant protein from the washed inclusion bodies was performed under denaturing condition by affinity chromatography using a Ni2+ Sepharose column (HistrapFF, GE HealthCare) following the manufacturer’s instructions.\n\nGradient elution was carried out by adding 5 volumes of imidazole at concentrations of 50 and 100 mM, and 10 volumes at 500 mM.\n\nThe collected samples were concentrated by ultrafiltration (Vivaspin 20, 10 kDa NMWL, GE HealthCare) and guanidine hydrochloride was removed by ethanol precipitation [15] before SDS-PAGE analysis [14] of eluted samples.\n\nProtein Analysis\nThe pellets from 1 mL samples of cells expressing rSpaA were re-suspended in 0.9 % (w/v) NaCl solution to an optical density (at 600 nm) of 3.0.\n\nFor SDS-PAGE analysis, the cell suspensions were mixed with 1 volume of sample buffer (0.125 M Tris, 10 % β-mercaptoethanol, 4 % SDS, 20 % glycerol, and 0.004 % bromophenol blue) and heated for 10 min at 95 °C.\n\n15 % SDS-PAGE was then performed with 20 μL of total protein samples in a Mini-Protean 3 Cell (Bio-Rad) run for 70 min at 200 V. The gels were stained with Coomassie Brilliant Blue R250, and de-stained with 10 % methanol and 10 % acetic acid.\n\nThe gels were photographed to estimate protein production, using the software ImageJ [16].\n\nBSA samples containing known concentrations (0.1, 0.2, 0.4, 0.8, and 1.0 mg mL−1) were used as standards to estimate the rSpaA concentrations in the gels.\n\nPreparation of Anti-rSpaA Antibodies\nAnti-rSpaA polyclonal antibodies were raised in mice against His-tagged rSpaA purified protein.\n\nOne animal was subcutaneously inoculated with 50 μg of protein mixed with Freund’s complete adjuvant.\n\nAfter 45 days, the animal received a second inoculation with 50 μg of protein mixed with Freund’s incomplete adjuvant, and after 10 days the immunized animal was bled, and the collected blood was centrifuged for 10 min at 4,000 rpm.\n\nThe supernatant was transferred to another tube, and centrifuged for an additional 3 min.\n\nThe final supernatant was recovered, and sodium azide solution was added to a final concentration of 0.02 %.\n\nWestern Blot\nCulture extract proteins from the SDS-PAGE gels were transferred to nitrocellulose membranes (HyBond ECL, Amersham Biosciences) at 150 V for 2 h. The membranes were blocked overnight at 4 °C with PBS-skim milk (5 %), and then washed three times (5 min each) with PBS-0.05 % Tween 20.\n\nA primary mouse anti-rSpaA antibody, diluted 1:500 in PBS buffer, or the mouse anti-HisTag antibody (Cell Signaling), diluted 1:10,000 in PBS, was applied to the membrane, followed by incubation for 2 h at room temperature with mild agitation.\n\nThe membranes were washed as described above, and incubated with anti-mouse IgG-AP linked antibody (Cell Signaling), diluted 1:3,000 in PBS for 1.5 h. After washing, the membranes were revealed using an AP Conjugate Substrate kit (Bio-Rad).\n\nResults and Discussion\nPartial ORF Cloning, Heterologous Expression, and Purification of the Antigenic SpaA Fragment from E. rhusiopathiae NCTC11002\nThe spaA gene fragment was successfully amplified from the genomic DNA of E. rhusiopathiae NCTC11002, as can be seen in Fig.\n\n2.\n\nThe amplified 1,026-pb fragment contains the region coding for the antigenic portion of the SpaA antigen [4, 5].\n\nDNA sequencing showed that this amplified fragment from the NCTC11002 strain had a high level of identity with the corresponding sequence from the Fujisawa strain, available on databases.\n\nThis is in agreement with previous reports that have shown that the spaA gene sequence is well conserved among many E. rhusiopathiae strains [4].\n\nThe recombinant protein was over-expressed under lacUV5 promoter control using the pET system and IPTG or lactose as inducer.\n\nThe main fraction of rSpaA produced was found as insoluble aggregates in the E. coli cytoplasm, and its purification was performed under denaturing conditions.\n\nAfter solubilization of the inclusion bodies using guanidine hydrochloride as denaturing agent, the rSpaA protein was purified by a single affinity chromatography step using a Ni-Sepharose column.\n\nHigh purity was achieved by imidazole gradient elution, as shown in Fig.\n\n3.\n\nEndotoxin content was not determined.\n\nThe predicted molecular weight of the recombinant protein was around 42 kDa.\n\nIn a Western blot analysis (Fig.\n\n4), the rSpaA showed a positive reaction with mouse anti-HisTag antibodies, as well as with mouse anti-rSpaA antibodies.\n\nThis demonstrated that the antibodies produced were able to recognize the recombinant protein.\n\nGrowth, Metabolite Formation, and Protein Production Under Auto-Induction Conditions\nThe data acquired from the flask cultures indicated that the levels of biomass and heterologous protein production differed according to the process strategy employed.\n\nFigure 5 illustrates the results obtained for cellular growth, substrate consumption, and metabolite formation.\n\nSimilar growth profiles were observed for conditions M1, M3, and M4, where induction was achieved by adding IPTG or lactose after an optical density of around 5.0 was reached (Fig.\n\n5b).\n\nUnder these conditions, cellular growth continued after induction as the cells were not exposed to a prolonged induction regimen.\n\nThe initial carbon sources were exhausted after between 8 and 12 h of cultivation, in all experiments (Fig.\n\n5a).\n\nFor conditions M1 and M4, previously accumulated acetate was consumed after exhaustion of the main carbon sources.\n\nTogether with other nutrients (from yeast extract and peptone), this enabled biomass formation to be sustained until the end of the process.\n\nFor condition M3, lactose added to promote induction (t = 8 h) was able to sustain cellular growth, and acetate produced during the first few hours remained in the cultivation medium (Fig.\n\n5c).\n\nIn the auto-induction experiment (M2), biomass formation was affected by the early induction imposed on the cells.\n\nThis prolonged stress condition caused slower growth with increasing amounts of acetate being formed throughout the cultivation.\n\nOn the other hand, as can be seen in Table 1, in the M2, protein production was not severely affected by the acetate accumulated, and a maximum specific production of 171 mgprot g −1DCW was reached in a short period of time (after 8 h of cultivation, which was the time of initial induction for the other conditions).\n\nThe results also showed that conditions where lactose was used as inducer (M2 and M3) produced more rSpaA than those with IPTG addition.\n\nA reasonable explanation for this lies in the fact that lactose, in addition to its inducer activity, also serves as a source of carbon and energy.\n\nIn addition, in experiments where IPTG was used as inducer, the cells experienced nutrient starvation, affecting protein expression.\n\nFigure 6 shows the recombinant protein production profiles according to time for the different strategies employed.\n\nUnder the auto-induction conditions (M2), production of the rSpaA protein occurred before 8 h of cultivation, as can be seen in Fig.\n\n6.\n\nSimilar rSpaA-specific productions were achieved at the end of cultivations for conditions where the ZYM-5052 medium was employed (M2, M3, and M4), and the highest protein specific production values were obtained when lactose was used to induce protein expression (M2 and M3).\n\nComparing conditions M2 and M3, the auto-induction strategy (M2) had the advantage of faster protein accumulation, shortening the cultivation process.\n\nFor conditions where IPTG was employed as inducer (M1 and M4), the lower rSpaA production in the M1 experiments indicated that the well-balanced ZYM-5052 medium was superior to the modified LB medium for attainment of high heterologous protein expression.\n\nThe auto-induction condition resulted in an efficient process: rSpaA-specific production was the highest among the conditions evaluated, and its expression occurred simultaneously with cell growth.\n\nBy this approach, the cellular concentration reached an OD of 5.0 within 8 h of cultivation with a protein-specific production of 171 mgprot/gDCW, corresponding to around 25 % of the total cellular protein content.\n\nIn addition, an efficient purification protocol for this antigen candidate was developed by a single chromatographic step under denaturing conditions.\n\nBased on these results, further cultivation studies will be carried out as fed-batch bioreactor cultures with the objective of developing a production process for rSpaA as a potential candidate antigen for a subunit vaccine against swine erysipelas.\n\n----------\nAs a professional microbiologist, create a concise and comprehensive Chinese scientific report on Erysipelothrix Spp. research based on the 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