Amplification and cloning of the S100A7 gene

Amplification of the S100A7 gene by PCR (Figure 1a) and nested PCR (Figure 1b) was confirmed in 1.0% agarose gel (w/v) electrophoresis by detection of the expected 306 bp fragment (Figure 1). The fragment corresponding to gene S100A7 was sub-cloned in an expression vector (pAE/S100A7) after cleavage with the restriction enzymes Xho I and Hind III. The correct insertion, identity and integrity of gene S100A7 were demonstrated on the basis of DNA sequencing which presented an identical sequence to that in GenBank Accession number 002963 (data not shown). The recombinant plasmid (pAE/S100A7) was then introduced into E. coli BL21::DE3 for the expression of recombinant S100A7 (His-tag) protein.

Expression and purification of the recombinant S100A7 (His-tag) protein

The expression of rS100A7 (His-tag) protein fused with His-tag was carried out in E. coli BL21DE3. The highest amount of recombinant proteins was obtained when the IPTG inducer was not added to the culture medium. The rS100A7 (His-tag) protein was present in inclusion bodies which were denatured in 8 M urea before purification with the Ni-NTA affinity column under denaturing conditions. The yield of rS100A7 (His-tag) protein was 2.0 to 3.5 mg/mL culture medium. Purified rS100A7 (His-tag) protein (200 ng) was homogeneous by 12.5% SDS-PAGE and Western blotting using the anti-S100A7 (His-tag) polyclonal antibody described here (Figure 2).

Identification of rS100A7 (His-tag) protein by mass spectrometry

We did not try to remove the nonapeptide from rS100A7 (His-tag). The rS100A7 (His-Tag) protein was purified by SDS-PAGE, treated with trypsin and the hydrolysate analyzed by MALDI-TOF-MS (Figure 3a). Ions with the highest intensity were sequenced by CID-MS/MS. The peptides identified by sequencing accounted for 55% of the amino acid sequence of the S100A7 protein acquisition number P31151, Swiss-Prot data base (Figure 3b).

The protein rS100A7 (His-Tag) described here has a nonapeptide, MHHHHHHLE, attached to its amino terminus. The molecular mass of the intact protein determined by linear MALDI-TOF-MS (Figure 3c) was 12,655.91 Da compared to a calculated molecular mass of 12,653.26 Da (delta = + 2.65 Da) (ExPasy Proteomics Server, http://expasy.org). We never had the rS100A7 protein without the His-tag.

Western blot analysis of rS100A7 (His-Tag)

The gel in Figure 2, lane 1, shows that only a primary component in the purified recombinant protein was detected by the antibody or by Coomassie Blue staining and that the mobility in SDS-PAGE corresponded to a mass of approximately 11.5 kDa. A second component in much lower concentration is visible at a higher molecular weight and may be an oligomer.

Immunohistochemical analysis of the anti-S100A7 (His-tag) polyclonal antibody using TMA

The tissues spotted on the TMA slide were immunostained with the anti-recombinant S100A7 (His-tag) rabbit serum. Both neoplastic and non-neoplastic squamous epithelium reacted with the polyclonal and commercial monoclonal S100A7 antibody. Human S100A7 protein was detected by the anti-recombinant S100A7 (His-tag) rabbit serum (Figure 4).

The images of immunohistochemical reactions for the target protein shown in Figure 4 illustrate the immunoreactivity of the polyclonal antibody: Figure 4a and 4b show the negative controls (non-immune serum or absence of secondary antibody). The anti-recombinant S100A7 (His-tag) rabbit serum was able to detect both neoplastic and non-neoplastic squamous epithelium at 1:12,000 dilution (Figure 4c-j). The staining score was 1+ to 4+ with exclusive cytoplasm staining (C-F) and both cytoplasm and nuclear staining (Figure 4g-j).

We have not systematically compared the protein expression of S100A7 between normal and neoplastic tissues or with the clinical and pathological characteristics in detail. However, a preliminary analysis of the results permitted us to determine variations in the levels and location of the protein in neoplastic epithelia obtained from HNSCC (Figure 4, panels g-j). The human S100A7 protein is produced in oral tissues, with cytoplasmic localization, during the early stages of the disease and its expression increases with disease progression (cytoplasmic and nuclear localization) (Figure 4, panels g-j), suggesting its association with the progression and recurrence of HNSCC [8].

Cloning the S100A7 gene

The open reading frame of the human S100A7 gene was obtained by PCR using a cDNA template from tracheal tumor tissue ("Projeto Genoma Clínico", FAPESP and Ludwig Institute for Cancer Research, Brazil) [13, 14]. Primers containing cleavage sites for the restriction enzyme Xho I (5' ATGGATCCCTCGAG ATGAGCAACACTCAAG 3') and Hind III (5' ATAAGCTT TCACTGGCTGCCCCCGGAA 3') were synthesized by Invitrogen (Carlsbad, CA, USA). For PCR, 10 μg from trachea tissue furnished by the human tissue cDNA bank of the Regional Center for Hemotherapy of Ribeirão Preto, University of São Paulo, was used. The reaction mixture contained 1 mM dNTPs, 7.5 mM sense and complementary primers, 1 U Platinum Taq DNA polymerase in 50 mM Tris-HCl, pH 8.8, containing 500 mM KCl, 5 mM MgCl2 (Invitrogen) and 1% Tween 20 (Sigma, Saint Louis, MO, USA). PCR consisted of an initial denaturation step of 5 min at 94°C, followed by 35 cycles of 40 s at 94°C (denaturation), 1 min of annealing at 53°C (the specific annealing temperature for the S100A7 gene) and 1 min at 72°C (extension). A second nested PCR assay was then performed using 1 μl of the reaction mixture described above and annealing at 60°C. The amplified 306 bp product was purified on 1.0% (w/v) agarose gel using the Wizard SV Gel, PCR Clean-Up System kit (Promega, Madison, WI, USA) and inserted into the propagation vector pCR2.1-TOPO using the TOPO-TA cloning kit (Invitrogen) according to the manufacturer's instructions. The construct was introduced into E. coli DH5α by the thermal shock method described by Sambrook and Russell [15] and the recombinants were selected on solid Luria-Bertani (LB) medium containing ampicillin (100 μg/ml). Plasmid DNA was extracted with the plasmidPrep Mini Spin kit (GE Healthcare, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's instructions. The structure of the construct was confirmed by nucleotide sequencing using the Thermocycle Sequencing BigDye Terminator kit (Applied Biosystems, Carlsbad, CA, USA) in an ABI Prism 377 automatic sequencer (Applied Biosystems). The S100A7 fragment was removed from the pCR2.1-TOPO vector by digestion with the restriction enzymes Xho I and Hind III and attached to the expression vector pAE, which contained the 6xHis-tag peptide in the N-terminal region [16], according to the protocol for the enzyme T4 DNA ligase (Promega). The construct was introduced into competent E. coli DH5α cells with the Subcloning Efficiency™ Chemically Competent E. coli kit (Invitrogen). The transformants were selected by PCR and plasmid DNA was extracted as described by Sambrook and Russell [15] and sequenced using the DYEnamic ET Dye Terminator Sequencing kit (GE Healthcare) with the automatic sequencer MegaBace™ 1000 (GE Healthcare). This indicated that the insertion of the 306 bp fragment corresponding to the S100A7 gene into the expression vector pAE was correct. The sequence of the fragment was identical to that deposited in GenBank as accession number 002963.

Expression of rS100A7 (His-tag) protein

The E. coli BL21::DE3 host was transformed with the vector pAE+S100A7 and plated onto selective medium. A single colony was used for the inoculation of 200 ml CircleGrow liquid medium containing 100 μg/mL ampicillin into a 500 ml Erlenmeyer flask. The culture was maintained at 37°C and stirred until it reached an absorbance of approximately 0.5 at 600 nm. Isopropyl-β-D-1-thiolgalactopyranoside (IPTG) at 1 or 10 mM was added to try to increase the amount of protein produced.

Purification of rS100A7 (His-tag) protein

E. coli BL21::DE3 cultivated in 500 ml of culture medium was centrifuged at 5,000 g for 10 min and the pellets were stored at -80°C. They were resuspended in 5 ml lysis buffer A (6 M GuHCl; 0.1 M NaH₂PO₄, 10 mM Tris-Cl; pH 8.0) and maintained at 4°C for one hour with shaking at 1,000 rpm. The lysate was centrifuged at 10,000 g for 30 min at 4°C (Eppendorf 5804 R) and 1.0 ml the supernatant was loaded onto an Ni-NTA SuperFlow affinity chromatography minicolumn equilibrated with buffer A (Qiagen, Hilden, Düsseldorf, Germany). The remainder of the lysate (4.0 ml) was processed at 1.0 ml/minicolumn. Each was centrifuged at 700 g for 2 min. After loading the minicolumn was washed with 600 μl buffer C (8 M urea; 0.1 M NaH₂PO₄, 10.0 mM Tris-Cl; pH 6.3) and centrifuged at 700 g for 2 min two times. The protein was eluted with 200 μl Buffer E (8 M urea; 0.1 M NaH₂PO₄, 10.0 mM Tris-Cl; pH 4.5) and centrifuged at 700 g for 2 min, three times.

The homogeneity of the recombinant S100A7 (His-tag) protein (10 μg) was evaluated by 12.5% SDS-PAGE [17]. The gel was stained with colloidal Coomassie blue G-250 (Serva, Heidelberg, Germany) according to Neuhoff et al. [18]. The amount of recombinant protein was measured with a DC Protein Assay kit (Bio-Rad) based on the method of Bradford [19] and determined by absorbance at 595 nm in a Versa-Max microplate reader (Molecular Devices, Sunnyvale, CA, USA).

Characterization of the recombinant S100A7 (His-tag) by mass spectrometry

Recombinant S100A7 (His-tag) protein was characterized after in situ trypsin digestion of the SDS-PAGE gel band corresponding to 11.5 kDa. The band was cut from the gel, destained with 0.1 M NH4HCO3 in 50% acetonitrile, dehydrated with 100% acetonitrile, dried in a SpeedVac (Savant Inc., Ramsey, MN, USA), and rehydrated with 20 μl 0.1 M NH₄HCO₃ containing 0.5 μg trypsin (Promega). The protein was digested at 37°C for 24 h. The hydrolysate was desalted in a microtip filled with POROS 50 R2 resin (PerSeptive Biosystems, Foster City, CA, US) and analyzed with a MALDI-TOF/TOF mass spectrometer (Axima Performance, Kratos-Shimadzu Biotech, Manchester, UK) equipped with a collision chamber for collision-induced dissociation (CID), with helium as the collision gas. The desalted hydrolysate was dissolved in 10 μl matrix solution containing 5 mg/ml alpha-cyano-4-hydroxycinnamic acid in 0.1% trifluoroacetic acid and 50% acetonitrile. Two microliters of the sample-matrix solution were applied to the stainless steel MALDI plate and allowed to evaporate. The mass spectrometer was calibrated with a mixture of angiotensin II, ACTH fragment 18-39, bradykinin fragment 1-7 and the oxidized B chain of bovine insulin (Sigma). The PMF and MS/MS spectra were analyzed with Mascot software V.2.2.4 (Matrix Science, London, UK) and compared with the structure of S100A7 (His-tag) construct.

The molecular weight of the intact rS100A7 (His-tag) protein was determined with MALDI-TOF/TOF-MS operated in the linear mode after desalting with POROS 50 R2. The instrument was calibrated with insulin, cytochrome C, chymotrypsin and bovine serum albumin.

Production of polyclonal antibody anti-rS100A7

The anti-recombinant S100A7 (His-tag) rabbit serum was obtained according to standard protocols [20]. Briefly, two adult female New Zealand white rabbits were immunized with 2.0 mg/animal of purified recombinant S100A7 (His-tag) protein emulsified in complete or incomplete Freund's adjuvant (Sigma) by subcutaneous or intramuscular injection, respectively. Before immunization, blood was obtained from each animal to prepare non-immune serum. The immunization efficiency was analyzed by Western blotting and serum samples were stored at -20°C.

Reaction of rabbit polyclonal antibody with recombinant S100A7 (His-tag) protein under denaturing and reducing conditions

Western blotting was performed after 200 ng of purified recombinant S100A7 (His-tag) protein was submitted to 12.5% SDS-PAGE [17] and the protein was transferred from the gel to a polyvinyldene fluoride (PVDF) membrane in buffer containing 25 mM Tris, 192 mM glycine, pH 8.3, and 10% methanol. A constant voltage of 35 V and 100 mA was applied. A PVDF membrane and the Chromogenic Western Blot Immunodetection kit (Invitrogen) were used according to manufacturer's instructions. The membranes were incubated separately with the primary antibodies, i.e., the anti-recombinant S100A7 (His-tag) rabbit serum at 1:5,000 dilution, and the positive control monoclonal antibody S100A7 (Novocastra, Newcastle Upon Tyne, UK) at 1:1,000 dilution. Finally, the membranes were washed in MilliQ water and incubated with the chromogenic substrate 5-bromo-4-chloro-3-indolyl-1-phosphatase (BCIP) and nitroblue tetrazolium (NBT). After protein labeling, the membranes were washed with MilliQ water in order to stop the reactions.

Interaction of the polyclonal antibody anti-rS100A7 with tissue microarray (TMA)

The anti-recombinant S100A7 (His-tag) rabbit serum was used for the detection of S100A7 by immunohistochemistry in tissue samples arranged in a TMA. Representative areas of tissues fixed in formalin and embedded in paraffin were selected randomly and used for the construction of a TMA block which contained up to four spots each of non-neoplastic tissue (Malpighian epithelium) and of neoplastic tissue (squamous cell carcinoma) from eight cases of neoplasias of the head and neck region (larynx). The TMA experiment was conducted using the polyclonal antibody at 1:2,000 to 1:16,000 dilutions. The anti-S100A7 monoclonal antibody (Novocastra) was used as a positive control at a dilution of 1:50 to 1:200. All dilutions were incubated at 4°C for 16 h after antigen recovery under the following conditions: incubation with 10 mM sodium citrate, pH 6.0, or incubation with 1 mM EDTA, pH 8.0, in a pressure cooker for 40 min, or in the absence of antigen recovery. The reaction was amplified using the NovoLink Polymer Detection System kit (Novocastra) according to manufacturer's instructions. The reaction was developed with the chromogenic substrate containing 0.10% diamine benzidine (Sigma, Saint Louis, MO, US), 0.06% hydrogen peroxide, and 1% dimethyl sulfoxide (Labsynth) in PBS buffer. Sections containing TMA were then counterstained with Harris hematoxylin and rapidly immersed in 0.5% ammonium hydroxide. Immunoreactivity was scored as follows: absent (no color) or positive (brownish red) present in up to 5% of the cells of interest (+1), present in > 5% and ≤ 25% of the cells of interest (+2), present in > 25% and ≤ 50% of the cells of interest (+3), and present in > 50% of the cells of interest (+4).

The controls were: positive - spots containing human lung - and the negative control was without the primary antibody.