Bacterial strains
The Escherichia coli strains NovaBlue (Novagen, Merck Millipore, Germany), SHuffle®T7 (C3026H, New England Biolabs, Germany) and Origami™ 2(DE3)pLysS (Novagen, Merck Millipore, Germany) were routinely grown in LB medium and maintained under standard conditions [37]. E. coli transformants were selected with 120 μg/mL ampicillin and 34 μg/mL chloramphenicol as indicated. Protein expression was carried out using EnPresso B medium [29-31] including booster tablets following protocols provided by the manufacturer (BioSilta Oy, Finland). Unless otherwise stated, chemicals and reagents were obtained from Sigma-Aldrich or Roth (Carl Roth GmbH & Co. KG, Germany).
Plasmids
Plasmid pET-23d was obtained from Merck Millipore (Germany) and plasmid pET23d(+)::HisDap was constructed encoding a diaminopeptidase-cleavable histidine-tag [37]. DNA was prepared using the Roti®-Prep Plasmid MINI kit (Carl Roth GmbH & Co. KG, Germany) and DNA fragments were isolated from agarose gel blocks employing the QIAquick Gel Extraction Kit (Qiagen, Germany). Plasmid pMJS9 [16] was kindly provided by Prof. Dr. L. W. Ruddock.
A gene sequence was designed to express amino acids 52–571 [8] of the human N-acetylgalactosaminyltransferase 2 [GalNT2, GeneID:2590, UniProtID:Q10471] and the restriction sites NdeI and XhoI were added at the 5′ and 3′ end of the DNA fragment, respectively. As a result, an additional methionine residue followed by an alanine moiety was added to the N-terminal sequence. The codon usage of the gene fragment was adapted for expression in an Escherichia coli strain background [38]. The synthesised DNA fragment was purchased from GeneArt® Gene Synthesis and supplied as plasmid construct cloned into the pMK-RQ shuffle vector (Life Technologies GmbH, Germany). Plasmid-DNA was digested with NdeI and XhoI, the 1.8 kb fragment was isolated and ligated with vector pET23d(+)::HisDap treated with the same restriction enzymes. The DNA was subsequently used to transform E. coli NovaBlue. As a result, the decahistidine-tag with the sequence MAHHHHHQHQHQHQHQH was translationally fused to the N-terminus of the expressed protein thus facilitating purification of the product. The resulting construct pET23d(+)::HisDapGalNAcT2 was verified by restriction digests and the cloned fragment was confirmed by DNA sequence analysis.
Protein expression of HisDapGalNAcT2
Plasmid pET23d(+)::HisDapGalNAcT2 was first expressed in Origami™ 2(DE3)pLysS and the production of insoluble HisDapGalNAcT2 was detected. The bacterial suspension was lysed by using ultrasonic sound. Afterwards the whole cell lysate was centrifuged to separate the soluble fraction (supernatant) from the particular fraction (cell pellet). Whole cell lysate, supernatant and particulate fraction were analysed by SDS-PAGE. Subsequent experiments were carried out with E. coli SHuffle® T7 as strain background.
Plasmids pET23d(+)::HisDapGalNAcT2 and pMJS9 were used to transform and co-transform the E. coli host strain SHuffle® T7. Plasmid pMJS9 encodes the sulfhydryl oxidase Erv1p and the protein disulfide isomerase PDI under the control of the arabinose-promoter [16]. Transformants were grown on LB agar plates supplemented with the required antibiotics and a single colony was used to inoculate a 2 mL liquid culture. Following incubation, culture aliquots were frozen at −80°C employing the CRYOBANK™ system (CRYOBANK™, Mast Diagnostica GmbH, Germany). For expression experiments two ceramic beads were transferred from a cryovial into a 50 mL Falcon tube containing 2 mL LB media (120 μg/mL ampicillin, 34 μg/mL chloramphenicol and 0.2% glucose) and the cells were maintained at 37°C, 175 rpm for 8 h. The culture was transferred into a 2 L baffled flask containing 200 mL EnPresso B medium (120 μg/mL ampicillin, 34 μg/mL chloramphenicol, 100 μL Reagent A) and grown at 30°C, 175 rpm for 15 h. Booster solution was added following the manufacturer’s instructions. The pre-induction of the pMJS9 encoded gene products was carried out in the presence of 0.5% w/v arabinose. Isopropyl-β-D-thiogalactopyranosid (IPTG) was added after 30 min to a final concentration of 1 mM to induce expression of the glycosyltransferase HisDapGalNAcT2 and the culture was incubated at 30°C, 175 rpm for 23.5 h. The cells were harvested and 10 mL fractions were pelleted via centrifugation at 4°C, 5500 g for 5 min. The supernatants were discarded and the cell sediments stored at −80°C. A scaled-up version of this experiment was carried out and a 15 mL culture was prepared to inoculate 1.5 L expression medium in a 2 L bioreactor (BIOSTAT®Bplus, Sartorius Stedim Biotech, Germany) at 30°C, 60% pO2, 0.5 vvm, pH 7, adopting conditions and procedures already described. Cell pellets were pooled and stored at −80°C. A 7.98 g portion of the cell sediment was used for protein purification.
Protein purification
A cell pellet derived from a 10 mL fraction of the expression culture was resuspended in 1.26 mL extraction buffer (50 mM Tris, 300 mM NaCl; pH 8) containing 140 μL lysozyme, 3 μL DNAse and 50 μL protease inhibitor (complete protease inhibitor cocktail tablet, F. Hoffmann La-Roche AG, Switzerland). The bacterial suspension was cooled on ice for 30 min and sonicated for 3 min on ice. The cell lysate was centrifuged at 4°C, 16 100 g for 10 min and the supernatant was passed through a 0.45 μM filter. The polyhistidine-tagged protein HisDapGalNAcT2 was purified using Ni-NTA spin columns (Qiagen, Germany) with washing buffer (50 mM Tris, 300 mM NaCl, 20 mM imidazole) and elution buffer (50 mM Tris, 300 mM NaCl, 500 mM imidazole) adjusted to pH 8 prior to use.
Similarly, 29 mL lysate were generated using a 7.98 g portion of the previously isolated cell pellet. HisDapGalNAcT2 was purified on a HisTrap HP 1 mL column with an ÄKTA Purifier (GE Healthcare Life Sciences). The column was equilibrated with 8 column volumes (CV) equilibration buffer (50 mM Tris, 300 mM NaCl; pH 8) and the 29 mL sample was loaded, followed by 8 CV of washing buffer (50 mM Tris, 300 mM NaCl, 20 mM imidazole; pH 8). The protein was eluted (50 mM Tris, 300 mM NaCl, 500 mM imidazole; pH 8) using a linear gradient from 20 mM to 500 mM imidazole over 4 CV. 1 mL fractions were collected for further analysis.
Analysis of purified protein
SDS-PAGE and immunoblot analyses were carried out using standard protocols with dithiothreitol (DTT) as reducing agent. Densitometric analysis was performed employing Fusion-FX software (Vilber Lourmat, Germany). For Western blot analysis, proteins were transferred to polyvinylidene fluoride membranes (PVDF, Thermo Scientific Inc., USA) and treated with mouse anti human GALNT2 antibodies (H00002590-A01, Acris Antibody GmbH, Germany) or penta-His antibodies (34660, Qiagen, Germany). Binding was detected with horseradish peroxidase (HRP)-labeled secondary antibodies and chemiluminescence was assessed using Roti®Lumin solutions.
Purified proteins were analysed by size exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) and circular dichroism (CD) spectroscopy to determine molecular mass, size, aggregation state/dispersity, folding state and thermal transitions of the protein [23,24]. Proteins were separated according to different molecular masses using a size exclusion column (SEC) (Yarra SEC-2000, Phenomenex Inc., Germany) on a Multi-Angle static Light Scattering (MALS) detector (Dawn®8+ HELEOS, 663.9 nm) in combination with an ÄKTA Explorer (GE Healthcare Life Sciences) and protein concentrations of the fractions were measured (Optilab® T-rEX Refractive index detector, 658 nm, 25°C, and ASTRA® Software, Wyatt Technology Europe GmbH, Germany). 100 μL of the purified and filtrated samples (0.2 μm, polyethersulfone membrane) were injected at a concentration of 0.13 mg/mL in running buffer (50 mM Tris, 300 mM NaCl, pH 8.0). Flow rate was adjusted to 0.5 mL/min.
Spectra of a filtered solution (NanoSep® MF 0.2 mM, Pall) containing 0.2 mg/mL HisDapGalNAcT2 or rhGALNT2 in 20 mM sodium phosphate buffer (pH 7.3) were recorded in a 715 CD-spectropolarimeter (Jasco, Hachioji, Japan) at 25°C. Spectra were measured from 190 – 260 nm with wavelength steps of 0.1 nm and a scan speed of 50 nm per minute. The averaged signal from four scans was corrected for the buffer signal. Thermal transitions were recorded at 208 nm with a step size of 0.1°C and a thermal slope of 1°C per minute. The data was fitted using a Boltzmann equation \( y=\frac{{\mathrm{A}}_1\hbox{-} {\mathrm{A}}_2}{1+e\left(x-{x}_0\right)/dx}+{\mathrm{A}}_2 \) to obtain the transition point (OriginPro 8, OriginLab, Northampton, Massachusetts, USA).
Protein activity assay
The activity of HisDapGalNAcT2 was monitored using a glycosyltransferase activity kit (EA001, R&D Systems Europe Ltd., United Kingdom) based on the release of phosphate during transfer of glycosyl residues to an acceptor peptide. UDP-N-acetylgalactosamine was used as donor substrate in combination with the acceptor substrates mucin EA2 (Eurogentec SA Anaspec, Belgium) comprising the peptide sequence PTTDSTTPAPTTK [10] or Filgrastim (Neupogen®, Amgen Inc.). Prior to use, the elution buffer of the purified HisDapGalNAcT2 (50 mM Tris, 300 mM NaCl, 500 mM imidazole; pH 8) was exchanged by 25 mM Tris, 150 mM NaCl, pH 7.3. The protein sample was stored at 4–8°C for about 3 weeks. Control assays were carried out using purified rhGALNT2 isolated from murine myeloma NS0 cells (7507-GT-020, R&D Systems Europe Ltd., United Kingdom). The enzyme was provided by the manufacturer in 25 mM Tris, 150 mM NaCl, 0.05% w/v Brij-35; pH 7.5 and stored in the freezer at −80°C.
Samples with EA2 as acceptor substrate were desalted, concentrated (ZipTip®μC18, Millipore, USA) and embedded into a HCCA matrix (α-cyano-4-hydrosycinnamic acid) for subsequent MALDI-TOF-MS analysis. Filgrastim samples were digested with trypsin prior to Electrospray Ionisation Mass Spectrometry (ESI-MS). Both analyses were carried out at the life science center of the University of Hohenheim (Germany).