Figure 1.
Determination of TGF-β1 in preparations of recombinant fibrillin-1 fragments. A:
Schematic overview of recombinant fibrillin-1 fragments used in this study. B: A sandwich ELISA was used to measure levels of active (grey bars) and total (black bars) human TGF-β1 present in samples of purified recombinant fibrillin-1 fragments. In order to activate latent LAP-TGF-β1 into immunoreactive active TGF-β1, the fragments were acidified with 1 M HCl for 10 min (see Materials and Methods). This enabled the measurement of total TGF-β1 (active and latent) in purified samples of recombinant fibrillin-1 proteins. A typical result is shown. The experiment was carried out in triplicates on the same microtiter plate. Standard deviations are indicated. The coefficient of determination (r2) for the regression analysis of the standards used was 0.99. C: Cell signaling assay. The recombinant fragments were diluted to 0.4 µM in 0.2 ml of serum-free medium (SFM) and added onto MSU 1.1 cells for 1 h at 37°C. The cell lysate was analyzed by Western blotting for phospho-Smad2 (pSmad2). 4 nM of recombinant TGF-β1 (rTGF-β1) was used as a positive control, and SFM alone was included as a negative control. Total Smad2/3 and GAPDH were used as loading controls. Smad2 migrates at 60 kDa (upper band), and Smad3 migrates at 52 kDa (lower band). The positions of globular marker proteins are indicated in kDa. Note that all recombinant fragments, except rFBN1-C, trigger TGF-β1 signaling. Data in panels B and C do not correspond to the same batches of recombinant proteins. A typical result is shown.
Figure 2.
Mock and rF20 purification by IMAC. A:
Chromatogram illustrating the elution profile of concentrated medium from non-transfected HEK293 cells on the IMAC column. The elution volume is indicated on the x-axis and the absorbance at 280 nm is indicated on the left-axis (black line). Protein bound to the column was displaced by an imidazole gradient as indicated (dashed line). B: Chromatogram illustrating the elution profile of rF20 on an IMAC column. C: Aliquots (20 µl) of relevant peak fractions from the mock purification were analyzed on a 7.5% SDS-PAGE and Coomassie staining (SM = Start Material, FT = flow-through). Pool 1 corresponds to weakly/non-specifically bound proteins. Pool 2 corresponds to the elution volume correlating with the elution volume for rF20 (see D, Pool 2). As the medium was from non-transfected HEK293 cells no protein peak at OD280 nm occurred in the absence of a strongly expressed recombinant protein. D: Aliquots (20 µl) from relevant peak fractions were analyzed from the rF20 purification on a 7.5% SDS-PAGE and Coomassie staining. E: 20 µl of Pool 2 from the mock and the rF20 purification visualized by colloidal Coomassie staining on a 7.5% gel under reducing conditions. Pool 2 rF20 was overloaded in order to identify minor bands. Four bands labeled (1), (2), (3) and (4) were excised and analyzed by MS/MS. They are identified as tenascin C (1), keratin-1 (2) keratin-9 (3) and fibrillin-1 (4). M indicates globular marker proteins in kDa. F: The concentrations of TGF-β1 in ng/ml present at every step of the purification scheme was determined by an ELISA (r2 = 0.982). Conditioned medium refers to medium collected from confluent cell layers. Concentrated medium is the concentrate of the ultrafiltration that is loaded onto the IMAC column. Plain bars represent active TGF-β1 present in the mock (white) and rF20 (light grey) purification at different stages. The dashed bars illustrate total TGF-β1 (active and latent) present in the mock (white) and rF20 (dark grey) purifications. The threshold level for reliable readouts is indicated by a dashed line.
Figure 3.
Cell signaling assay with Pool 2 of the mock purification.
50 µl of Pool 2 of the mock purification (see Fig. 2A) was titrated two-fold by serial dilutions in 0.2 ml of SFM, and added on MSU 1.1 cells for 1 h at 37°C. LAP-TGF-β1 was activated (+) by acidification (1 M HCl, 10 min). Concentrations of active and total TGF-β1 are indicated in pM. The negative control is 50 µl of TBS/Ca treated with 1 M HCl for 10 min and diluted in 0.2 ml of SFM. 4 nM rTGF-β1 diluted in SFM is included as the positive control. The cell lysate was analyzed by Western blotting for pSmad2 (12 µg of total protein), total Smad2/3 (12 µg of total protein), and GAPDH (5 µg total protein); the latter two are loading controls. The positions of the globular marker proteins are indicated in kDa.
Figure 4.
Pre-absorption chromatography.
Concentrated medium containing rFBN1-N was loaded onto an IMAC column without pre-loaded nickel ions. A: The chromatogram displays the purification profile of rFBN1-N on the empty IMAC column. The left-axis (black line) displays absorbance at 280 nm. The x-axis denotes elution volume and right-axis (dashed black line) indicates the imidazole gradient. Pool 1 (P1, 20–22 ml) and Pool 2 (P2, 25–27 ml) indicate the volume (ml) at which protein contaminants and homogenous recombinant protein elute respectively during a standard IMAC purification of rFBN1-N. B: Aliquots (20 µl) from relevant peak fractions were analyzed from the rFBN1-N purification in A on a 7.5% SDS-PAGE under reducing conditions and stained with Coomassie Brilliant Blue. SM = start material, FT = flow-through, and M indicates globular marker proteins in kDa. C: TGF-β1 sandwich ELISA with fractions from the Pool 1 and Pool 2 of pre-absorption chromatography (r2 = 0.991). The experiment was carried out on the same microtiter plate. The threshold level for reliable readouts is indicated by a dashed line. The data represents mean of duplicates, and standard deviations are indicated.
Figure 5.
TGF-β1 antibody affinity chromatography of rF20.
Recombinant rF20 was subjected to antibody affinity chromatography using an immobilized anti-TGF-β1 antibody (see Materials and Methods). A: The chromatogram illustrates the elution volume (x-axis, ml) versus the absorbance measured at 280 nm (left-axis, black line). TGF-β1 is eluted from the column with 0.1 M glycine-HCl, pH3 (right-axis, dashed line). The inset demonstrates aliquots (20 µl) of representative peak fractions separated on a 4–12% SDS-PAGE under reducing conditions, and analyzed by AgNO3 staining. Positions of globular marker proteins (M) are indicated in kDa; SM = start material. B: A sandwich ELISA was used to assess levels of TGF-β1 present in the rF20 fractions before and after antibody affinity chromatography. The r2 for the standard curve was 0.99. Latent LAP-TGF-β1 was activated by acidification in to active TGF-β1. This enabled the measurement of total TGF-β1 (active and latent) in the start material (SM) and eluted fractions (ml). The threshold level for reliable readouts is indicated by a dashed line. The experiment was carried out in duplicates on the same microtiter plate. Standard deviations are indicated. C: Cell signaling assay. 50 µg/ml (0.4 µM) of standard-purified (IMAC) and in addition affinity-purified rF20 was added onto HSFs for 1 h at 37°C. LAP-TGF-β1 was activated (+) into active TGF-β1 by acidification. Buffer control corresponds to TBS/Ca diluted in SFM. 4 nM of rTGF-β1 is included as the positive control. The cell lysate was analyzed by Western blotting for pSmad2 (12 µg total protein), total Smad2/3 (12 µg total protein), and GAPDH (5 µg total protein). The positions of the globular marker proteins are indicated in kDa.
Figure 6.
Gel filtration chromatography of rFBN1-N and rF23.
Fibrillin-1 fragments rFBN1-N and rF23 were subjected to gel filtration chromatography on an analytical Superose 12 column equilibrated with 50 mM Tris-HCl, 800 mM NaCl, pH7.4. A: TGF-β1 present in the rFBN1-N preparation before and after gel filtration was quantified by a sandwich ELISA. The r2 for the standard curve was 0.981. To measure total TGF-β1 in the start material (SM) and eluted fractions (ml), latent LAP-TGF-β1 was activated by acidification into immunoreactive active TGF-β1. The experiment was carried out in triplicates on the same microtiter plate. Standard deviations are indicated. The threshold level for reliable readouts is indicated by a dashed line. The inset illustrates the elution profile of rFBN1-N at OD280 nm. The major protein peak is at 8 ml while the peak at 17 ml reflects protein trailing, which is typical for recombinant fragments encompassing the N-terminal half of fibrillin-1. B: Cell signaling assay. HSFs were incubated with 0.4 µM (56.4 µg/ml) of rFBN1-N prior to and after gel filtration chromatography for 1 h at 37°C. LAP-TGF-β1 was activated (+) into active TGF-β1 by acidification. The buffer control refers to TBS/Ca treated with 1 M HCl for 10 min, and diluted in 0.2 ml of SFM. 4 nM of rTGF-β1 is included as the positive control. The cell lysate was analyzed by Western blotting for pSmad2 (12 µg of total protein), total Smad2/3 (12 µg of total protein), and GAPDH (5 µg of total protein). C: The ELISA results quantify TGF-β1 amounts present in the rF23 preparation before (start material, SM) and after gel filtration (represented by elution volume, ml). Experiment carried out in triplicates, and standard deviations are indicated (r2 = 0.984). The inset exhibits the elution profile of rF23. The major protein peak is at 12.5 ml while the peak at 17.8 ml reflects protein trailing. D: Cell signaling assay. HSFs were incubated with 0.4 µM (20 µg/ml) of the start material and the gel filtration purified rF23 for 1 h at 37°C.
Figure 7.
IMAC of recombinant TGF-β1 and LAP.
A: 1.5 µg of active TGF-β1 was loaded onto a 1 ml nickel-charged IMAC column. The x-axis denotes the elution volume (ml), and the left-axis denotes the amount of active TGF-β1. The right-axis (dashed line) illustrates the imidazole gradient. No protein peak was detected at OD280 nm (data not shown). The grey bars indicates active TGF-β1 (ng) present in the start material (SM), flow-through (FT) and eluted fractions (volume, ml), determined by a sandwich ELISA for TGF-β1. The experiment was carried out in triplicates, and standard deviations are indicated (r2 = 0.99). B: 5 µg of LAP (TGF-β1) was loaded onto a 1 ml nickel-charged IMAC column. The grey bars indicate the amount of LAP (ng) detected in the SM, FT and eluted fractions by a direct ELISA. The data represents mean of duplicates and standard deviations are indicated (r2 = 0.99).