Oligonucleotides

All pair of primers (Forward/Backward) used are listed below. Restriction enzyme sites, incorporated to facilitate cloning, are underlined and indicated in parentheses.

F-α: 5΄-ATG GTG CTG TCT CCT GCC GAC-3΄, B-α: 5΄-ACG GTA TTT GGA GGT CAG CAC GGT-3΄, F-TAT-α: 5΄-GCA GGT TCA TAT GCG CAA GAA ACG CCG CCA GCG CCG CCG CAT GGT GCT GTC TCC T-3΄ (NdeI), B-α-HA: 5΄-CAA CTC GAG TCA AGC ATA GTC TAA GAC GTC ATA ATA ACG GTA TTT GGA GGT CAG-3΄ (XhoI), F-α2: 5΄- GCA GGT TCA TAT GGT GCT GTC TCC TGC CGA CAA-3΄ (NdeI).

Construction of recombinant vectors

The cloning of the α-globin coding sequence (CDS) fused to the nucleotide sequence of the TAT peptide and the Hemagglutinin (HA) tag was carried out in both pCRII-TOPO vector (TOPO® TA Cloning® Kit with pCRII® TOPO®, Thermo Fisher Scientific, Massachusetts, United States) and the bacterial fusion expression vector pET-16b (Novagen, Darmstadt, Germany) [19].

Using human placenta -DNase treated- total RNA as a template and primer pairs F-α / B-α the α-globin CDS [Homo sapiens hemoglobin, alpha 1 (HBA1), mRNA NCBI Reference Sequence: NM_000558.4] was amplified using DreamTaq™ Hot Start DNA Polymerase (Thermo Fisher Scientific, Massachusetts, United States). The PCR product (429 bps) was ligated to pCRII-TOPO vector. Using this plasmid, as a template, and the primer pairs F-TAT-α-ΗΑ/B-α-HA and F-α2/B-α-HA, the TAT-α-globin-HA (485 bps) and α-globin-HA gene fragments (454 bps), respectively, were amplified. The two PCR products developed were then ligated to pCRII-TOPO. These plasmids were then digested and proceeded in «sticky ends» cloning, with the corresponding restriction enzymes, into pET-16b to generate the recombinant plasmids. Freshly prepared competent E. coli strain TOP10F' were transformed with the recombinant prokaryotic plasmids and plasmid DNA was isolated (Nucleospin Plasmid Kit, Macherey–Nagel, Düren, Germany). Clones containing the correct construct-inserts were selected via RFLP analysis and verified by automatic sequence analysis (CeMIA SA, Larissa, Greece).

Expression, purification and analysis of recombinant proteins

Escherichia coli strain CD43 (DE3) was transformed by the pET-16b-recombinant prokaryotic plasmids to express 10xHis-XaSITE-α-globin-HA and 10xHis-XaSITE-TAT-α-globin-HA proteins, as previously described [17,18,19]. The fusion proteins were purified by affinity Ni2+-NTA column chromatography in order to be used in the Κ-562 transduction experiments, in the in vitro assembly of α- and β-monomers and in the size exclusion HPLC experiments. For the affinity Ni2+-NTA column chromatography, HisPur™ Ni–NTA Resin agarose beads (Thermo Fisher Scientific, Massachusetts, United States) were used and the recombinant proteins were eluted from the column by adding gradient imidazole concertation buffers (containing 10 mM—400 mM imidazole) and then filtrated using a stirred ultrafiltration cell (Millipore, Massachusetts, United States) for purification.

Alternatively, bacterial pellets were collected and processed for isolation and purification of bacterial Inclusion Bodies (bacterial-IBs), dissolved in 1 M l-Arginine (l-Arg) Sigma-Aldrich, St. Louis, Missouri, United States) [17, 19] and further used in the transduction experiments, as in our previous works [17,18,19].

SDS − PAGE and Western Blot immunostaining analysis were carried out, as previously described [17]. The resolved proteins were separated in 14–15% SDS-PAGE and blotted with the mouse monoclonal anti-His.IgG (1:2600) (Sigma-Aldrich, St. Louis, Missouri, United States), the mouse monoclonal anti-HA-(antihemagglutinin).IgG (Santa Cruz Biotechnology Inc. California, United States) and rabbit polyclonal anti-GAPDH antibody (1:4000) (Flarebio Biotech LLC, New Jersey, United States). The membrane was then incubated with alkaline phosphatase-conjugated goat anti-mouse.IgG-AP (1:1000) (Santa Cruz Biotechnology Inc. California, United States) and goat anti-rabbit.IgG-AP (1:2500) (Sigma-Aldrich, St. Louis, Missouri, United States). Proteins were visualized by using NBT/BCIP (Biotium, California, United States) substrates for alkaline phosphatase.

LC − MS/MS technology for recombinant protein identification

Bacterial-IBs, enriched in recombinant 10xHis-XaSITE-TAT-α-globin-HA, as well as soluble (purified by Ni2+-NTA chromatography) 10xHis-XaSITE-α-globin-HA and 10xHis-XaSITE-TAT-β-globin-HA (used for in vitro assembly/formation into α-/β-globin tetramer analyzed by size exclusion HPLC) were subjected in LC − MS/MS analysis for protein sequence identification (Additional file 1: Methods).

In vitro assembly of α- and β- monomers and analysis by size exclusion HPLC

The in vitro assembly of α- and β- monomers into α/β-globin tetramers was evaluated when equal quantities of the recombinant 10xHis-XaSITE-α-globin-HA protein (∼40 μg) and of the 10xHis-XaSITE-TAT-β-globin-HA [19] (both, solely, purified by affinity Ni2+-NTA column chromatography) were mixed and incubated at R/T (Room Temperature) for different timed-intervals. The analysis of the reaction mixture containing the monomers of the recombinant α- and β-chains was achieved by size exclusion chromatography (SEC) at t = 0, 24 and 48 h post-incubation. Prior to incubation, both the recombinant proteins were incubated with 10 mM DTT for 1 h to reduce globin-dimers in the corresponding sample monomers [25]. Also, soluble recombinant proteins were separately, in different runs, analyzed by LC–MS/MS, as previously described, to identify the monomers’ protein sequence.

The SEC-HPLC system comprised of an Agilent HP 1100 series pump (Agilent, California, United States) multiple wavelength detector set at 270 nm and a Phenomemex BIOSEP-SEC-S3000 column (300 × 7.8 mm, 5 μm, Phenomenex, California, United States). The mobile phase was phosphate buffer 50 mM pH 6.8, containing 0.3 Μ ΝaCl and the flow rate was 0.5 mL min−1.

Cell culture and transduction experiments

Human Chronic Myelogenous Leukemia K-562 cells [26] were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics/antimycotic (Gibco-Invitrogen, Life Technologies Inc., Texas, United States) at 37 °C, in 5% CO2. For the transduction of TAT fusion protein, the cells were cultured to 70–80% confluence. Just before transduction, the culture media was removed and replaced by Opti-MEM I reduced Serum Medium (Gibco-Invitrogen, Life Technologies Inc., Texas, United States), to which the recombinant protein 10xHis-XaSITE-TAT-α-globin-HA was then added. K-562 cells were treated with 50 μg ml−1 soluble protein for indicated intervals (30 min and 1 h), in order to evaluate its transduction efficiency.

RBCs samples were derived from either peripheral blood or bone marrow of HbH donor patients (Patients 1–5), and all subjects gave written informed consent. RBCs were, also, cultured in RPMI-1640 medium supplemented with 10% FBS and antibiotics/antimycotic at 37 °C, in 5% CO2. Just before transduction, the culture media was removed and replaced by Opti-MEM, to which the recombinant protein 10xHis-XaSITE-TAT-α-globin-HA was then added (2 mg per 2 × 109 RBCs) in the form of bacterial IBs, dissolved in 1 M l-Arg, solution (pH 8.0). RBCs were incubated for 4 h and 48 h. A solution of 1 M l-Arg, pH 8.0, was used as control treatment. For incubation times longer than 2 h, 1 × volume of RPMI-1640 medium (supplemented with FBS and antibiotics/antimycotic) was added.

At timed-intervals during incubation, both K-562 cells and RBCs cells from Patients 1 – 3 and Patient 5, were harvested and washed twice with PBS 1 × (pH 7.4) buffer and processed for Western Blot analysis. Briefly, cells were lysed in standard RIPA lysis buffer, supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland) on ice for 15 min, centrifuged, collected as supernatants, and separated in a 14–15% SDS–polyacrylamide gel and immunoblotted, as described previously.

Evaluation of the effects of transduction of recombinant fusion α-globin chain on the phenotype of HbH RBCs inclusion bodies (HbH-IBs)

Exposure of RBCs to supravital staining dyes (such as methylene blue, methylene violet or brilliant cresyl blue) provides a helpful tool for the screening of RBCs to detect potential HbH patient clinical phenotype, where β4 tetramers appear as precipitated inclusions (HbH-IBs) [27]. RBCs from five HbH patients were treated with bacterial-IBs, enriched in 10xHis-XaSITE-TAT-α-globin-HA and washed twice with 1 × PBS. Subsequently, RBCs from four of them (Patients 1—4) were stained with methylene violet dye to evaluate the ratio of positively versus negative stained regarding the presence and/or absence of HbH-IBs, respectively.

HbH patients’ RBCs (more than 2 × 108 cells in 500 μl) were incubated, in a RIA vial, for 30 min, at 37 °C, with 50 μl of 0.018 M NaNO2. Two drops of the mixture were placed in a new RIA vial and stained with two drops of a staining dye, containing methylene violet 2B and potassium oxalate monohydrate, for 30 min, at R/T. After slide coating, the slides were left to dry for 30 min at R/T and then examined under a light microscope. The percentage of positive cells was calculated by counting more than 1000 cells per assay and by dividing the number of positive cells by the number of total cells and multiplying by 100, while negative cells were calculated via the equation [1.00 – (Number of negative cells ÷ Number of total cells)] × 100.

Statistical analysis

For the screening of HbH RBCs to evaluate the percentage of HbH-IBs, at least three independent biological repetitions from the four different HbH patients were performed. Statistical significance was achieved through an unpaired, parametric t test (p < 0.01 was the threshold for statistical significance) using Graph Pad Prism 6 (GraphPad Software San Diego, California, United States, http://www.graphpad.com).