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Basic techniques in molecular biology

Preparation of plasmid DNA

For transformation of E. coli XL-1 blue chemically competent cells, cells were thawed on ice for 5 mins. 50-100 ng plasmid DNA (or ~200 ng ligation product) were added, and cells were further incubated on ice for 20-30 mins. Afterwards, a heatshock was applied at 42°C for 45 secs. Cells were then further incubated on ice for 2 mins, and 950 μl of LB medium were added. Cells were then shaken at 200 rpm for at least 1 h at 37 C, and plated on LB-agar plates containing the appropriate antibiotic. The next day, single clones were picked and used to inoculate a liquid culture of 5 ml LB medium. After being incubated overnight shaking (200 rpm) at 37°C, cells were spun down, and plasmid DNA was extracted using the Qiagen QIAprep Spin Miniprep Kit. If ligation products were used for transformation, analytical digestion of DNA was performed by digestion with suitable restriction enyzmes for 1 h at 37°C and the correct incorporation of the desired fragment was verified via analytical gel electrophoresis. Additionally, plasmids were sequenced using the Eurofins Genomics sequencing service.

Determination of DNA concentrations

DNA concentrations were determined spectrophotometrically using a Nanodrop device. From the measured absorption at 260 nm, DNA concentrations were determined, an absorption of 1 being equal to a concentration 50 ng/μl DNA. Furthermore, wavelengths at 280 nm and 230 nm were measured to confirm sample purity without contamination of proteins (280 nm) or carbohydrates, phenol or EDTA (230 nm). Ratios of 260/280 nm being in the range between 1.6 and 2.0, as well as the ratios of 260/230 nm being between 2.0 and 2.2 generally indicate sample purity.

DNA digestion and ligation

For cloning, DNA was digested using restriction endonucleases. The amount of used enzyme hereby depended on the amount of to be digested DNA, whereas ~1 unit was used per μg of DNA. Buffers and DNA concentrations were used according to the manufacturer's suggestion. Digestions were incubated at 37°C. If deemed necessary, 5’-ends of vector fragments were dephosphorylated for 30 min at 37 °C directly by addition of alkaline phosphatase (Fast-AP) to the restriction mixture at 37°C.
Ligations were conducted using the appropriate DNA fragments while adding 400 units of ligase in the appropriate buffer. Vector and insert fragments were generally used at a 1:3 ratio, while the amount of vector fragment DNA was varied between 50-100 ng.

Agarose gel electrophoresis

For separation of DNA fragments, agarose gel electrophoresis was used. For fragments with a size of more than 500 bp, 1% (m/V) agarose gels were used. For smaller fragments, agarose concentration was increased as deemed necessary, until concentrations of up to 1.8% (m/V) agarose. For the production of gels, the appropriate amount of agarose was solved in 50 ml TAE buffer by heating the suspension. For staining, ethidium bromide was added into the mixture. Gels were run at 100 V for analytical and at 80 V for preparative use (200 mA respectively). For preparative gels, DNA fragments were excised and extracted from the gel using the Qiagen Gel Extraction Kit according to the manufacturer's protocol.

Polymerase chain reaction

For the amplification of DNA fragments, polymerase chain reaction was conducted. Hereby, 50 µl PCR mixture contained 50-500 ng template DNA, 100 pmol of each primer, and 10 nmol of each desoxynucleotide (dATP, dCTP, dGTP, dTTP). Additionally, the mixture contained 1 U Q5 high-fidelity polymerase and Q5 polymerase buffer, and was filled up with ddH₂O to the final volume. For the PCR, double-stranded fragments were initially denaturated at 98 °C for 2 min and subsequently amplified in 25-30 repetitive cycles, each comprising three steps: (1) Denaturation of the double-stranded DNA (98 °C, 30 s), (2) annealing of the primer to the single-stranded template for 30 s at 50-65 °C (depending on the melting temperature of the primer-template hybrid), and (3) extension of the primed DNA template (72 °C, 60 s per kb fragment length). After that, a final elongation step (2 min, 72 °C) was included to ensure that remaining single-stranded template molecules are fully elongated.

Then, the PCR product was separated by agarose gel electrophoresis to check for its correct size and isolated from the gel as described or directly isolated from the PCR reaction mixture, respectively using the Qiagen Gel extraction and PCR purification kit.

Oligonucleotide annealing

Preparation of chemically competent E. coli cells

A frozen glycerol stock of the desired strain of E. coli cells was streaked out on a LB plate under sterile conditions and incubated overnight at 37°C. A 4 ml LB medium liquid preculture was then inoculated and incubated overnight at 37°C shaking. 500 μl starter culture were used to inoculate a 50 ml LB medium liquid culture. When reaching an OD₆₀₀ of 0.45-0.5, the culture was immediately chilled on ice for 10 mins, and harvested by centrifugation at 3000g for 10 mins at 4°C. The supernatant was cast away, and the pellet resuspended in 40 ml of ice cold 100 mM MgCl₂. Cells were spun down again at 3000 g for 10 mins at 4°C, and the pellet resuspended in 20 ml of ice cold 50 mM CaCl₂. This suspension was kept on ice for 30 mins, and harvested by centrifugation at 3000 g for 10 mis at 4°C. The pellet was resuspended in 2 ml ice cold 50 mM CaCl₂ and 15% glycerol. The resulting bacterial suspension was sampled in 1.5 ml microcentrifuge tubes, frozen in liquid nitrogen and stored at -80°C until further use.

Basic techniques in protein biochemistry

Determination of protein concentration

Protein concentrations were determined spectrophotometrically. According to Lambert-Beer’s law, the measured absorption at 280 nm was normalised by the extinction coefficient of the protein as predicted by ProtParam based on its sequence.

Protein purification

After harvesting of cultures via centrifugation of liquid cultures at XX g for X mins, cells were lysed via X.

Immobilized metal ion affinity chromatography (IMAC)

Since the XX expression vector allows for the expression of the target protein as a SUMO/TEV peptide fusion construct with His₆-tag, the recombinantly expressed protein could be purified via IMAC. IMAC makes use of the interaction of imidazole side chains of histidine residues with immobilised nickel ions. Using this method, the target protein can be eluated by addition of imidazole, which competitively binds to the column and thus displaces the target protein in the column, causing it to eluate. After elution, the His₆-tag is being cut off using the SUMO protease in order to prevent interference in binding properties due to the tag. After centrifugation of samples, the resulting supernatant was purified using a stripped and equilibrated XX column (X ml volume). Therefore, the column was loaded with the supernatant and washed with buffer A until absorption hit zero. Then, the column was washed with wash buffer in order to remove competitively binding substances with weaker binding than the target protein. For elution, a -linear/step- elution gradient was set. The resulting fractions were analysed by SDS-polyacrylamide gel electrophoresis and were pooled for further purification, if their purity was deemed sufficient. (The protein concentration was measured and protease was added (please add concentration of protease here). In order to remove imidazole, dialysis was conducted overnight, using X l dialysis buffer.

Ion exchange chromatography (IEC)

Ion exchange chromatography is based on the separation of proteins with different electrostatic properties (pI values) by binding of proteins on a charged matrix and elution witha a salt gradient.

Size exclusion chromatography (SEC)

Size exclusion chromatography is a chromatographic method based on different diffusion times of proteins through a gel matrix in dependence on the hydrodynamic radius of the target protein; heavier proteins generally eluting earlier due to higher diffusion speeds through the gel matrix.

For size exclusion chromatography of X, Y ml of concentrate was loaded on a Z column. Resulting peak fractions were respectively loaded onto an SDS-gel and the fractions having an elution volume corresponding to the size of the target protein tetramer were, if deemed sufficiently pure, pooled and concentrated.

Discontinous SDS polyacrylamide gel electrophoresis (SDS-PAGE)

For analysis of recombinant protein expression, discontinuous SDS-polyacrylamide gel electrophoresis was conducted. Therefore, samples were mixed with Laemmli buffer (finalc concentration 1x) and incubated at 95°C for 5 min. For electrophoresis, currents of 300 mA were used, while voltage was initially set to X V and raised to X V after 10 mins. For staining, the gel was overlaid with staining solution and heated in the microwave. The staining solution was then replaced by XX.

Human cell culture

Seeding and passaging of HEK293T cells

Frozen HEK 293T cells, having been stored in liquid nitrogen, were quickly thawed in a 37°C water bath. Cells were then transferred into a tube containing pre-warmed Dulbecco’s Modified Eagle Medium medium (DMEM, including 10% fetal calf serum, 100 U/ml penicillin and 100 μg/ml streptomycin). After having been spun down (300 g, 5 mins), the supernatant was discarded and the pellet resuspended in DMEM. The cell suspension was then transferred into culturing flasks and incubated at 37°C and 5% CO₂ until further use. When reaching confluency, medium was removed from culture flasks and cells were washed twice with PBS. Cells were then treated with trypsine solution (0.05% in 1x PBS) and incubated at 37°C and 5% CO2 for 5 mins. After transferring cells into a 50 ml tube and adding 45 ml of medium, cells were counted and passaged into new culture vessels as deemed suitable.

Luciferase assay for the quantification of protein expression

For the quantification of expression of either the receptor or of nanoluciferase-containing constructs (e.g. for the choice of signal peptides and quantification of hypoxia-dependent promoter activity), luciferase assays were conducted.

Therefore, 4x10⁴ cells were seeded into 6-well plates in 4 ml DMEM medium until reaching the desired confluency. For transfection, 100 μl OptiMEM were mixed with 3 μg DNA and 12 μl Turbofect, vortexed for 10 s and incubated at room temperature for 20 mins. After removing 1 ml of supernatant, the transfection mixture was added drop-wise and the plate was incubated at 37°C and 8.5% CO₂ overnight. The next day, medium was removed and DMEM medium (10% FCS, Pen/Strep) was added.

For the luciferase assay, 50 μl of carefully removed medium were mixed with 50 μl luciferase assay buffer/sustrate mix from the NanoGlo Luciferase Assay Kit, and incubated for 10 mins at RT. Measurements were performed at 460 nm using a platereader.

Quantitative reverse transcription Polymerase chain reaction (RT-qPCR)

For the analysis of the gene expression of the different receptors on mRNA level RT-qPCR was performed.

Thus 1x10⁶ and 1x10⁵ cells were collected and spun down. The medium was discarded. QIAzol was added to the cell pellet and mRNA extraction was performed according to manufacturer's protocol (miRNeasy micro kit, QIAGEN). Afterwards mRNA concentration was measured by nanodrop. In addition the total mRNA was analysed for purity and integrity with an Bioanalyzer (Agilent Technologies). The measurement with a RNA Nano Chip was performed according to manufacturers protocol (Agilent technologies). To prepare the mRNA for quantitative analysis 750&n;g of each sample mRNA were used for reverse transcription (RT).

The mastermix for the RT reaction was mixed as following:

Three reverse transcriptase negative (Neg. RT) samples as well as two controles without template mRNA were added as negative controles. The RT was performed according to manufacturer's protocol (????).

The generated cDNA was then used for the quantitative PCR. The analysis was conducted on a Biorad CFX 1000 with 348 block using the SsoFast EvaGreen PCR mix.

Biophysical Methodology

Mass spectrometry

For confirmation of the molecular weight of proteins, electron spray ionisation-quadrupole time of flight was used. Protein masses were determined as a result from the mass/charge ratio (m/z). Herefore, 50-100 μl of a 0.1 mg/ml protein solution were dialyzed with 10 mM ammonium acetate (pH 6.6) overnight. For measurements, a mixture of 140 μl 10 mM ammonium acetate (pH 6.6), 40 μ acetonitrile, 20 μl of the dialized protein solution and 1 μl formic acid ( was injected.

Surface plasmon resonance spectroscopy

For the analysis of protein binding affinities and kinetics, surface plasmon resonance spectroscopy was used.

Fluorescence titration

Fluorescence titration is a method that utilizes the change of intrinsic protein fluorescence upon ligand binding (as mainly mediated by aromatic amino acid side chains) in order to quantify binding properties. This way, the binding affinity (as characterized by the K_D value) can be determined.

For the measurement, 2 ml of continuously stirred protein solution (1 μM) were titrated by the continuous addition of 100 μM D-biotin solution. For the measurements, temperatures of 20°C, an excitation wavelength of 295 nm and emission wavelengths of 350 nm were used (slid width of 5 mm, respectively) and the signal integrated over 5 s. The titrant was hereby added in 2 μl steps until a final titrant concentration of 2 μM.

Circular dichroism spectroscopy

For the analysis of protein folding states, circular dichroism spectroscopy was used, as it allows drawing conclusions about the formation of protein secondary structures.

For that purpose, three CD spectra were recorded, for each of which a 10 μM protein concentration according to the respective monomer and a cuvette with a 1 mm thickness was used. First, a UV-CD spectrum at 20°C was recorded with a start wavelength of 250 nm and an end wavelength 190 nm. Second a denaturation spectrum and third, a renaturation spectrum was recorded, both of which were recorded at 213 nm between 20 and 95 °C. As an exception, enhanced monomeric avidin was measured at 203 nm.