Materials
A gene of hFasLECD (amino acid residues, 139–281) containing double substitution mutations (N184Q and N250Q) with an N-terminal FLAG-(LysLysLysGlyCysGlyGlyGlyGly) tag sequence (NFK3G1CG4-hFasLECD) was constructed by introducing nine nucleotide bases (AAGAAGAAG) insertion mutation into the gene of NFG1CG4-hFasLECD. The production of NFK3G1CG4-hFasLECD in a P. pastoris secretory expression system was conducted as described previously [19]. hFasRECD-Fc was produced in a baculovirus – Bombyx mori expression system and purified as described in the previous paper [36]. Avidin from egg white (for biochemistry), normal rabbit IgG whole molecule (purified by Protein A), Pepsin from porcine stomach, 2-aminoethanethiol hydrochloride salt and washing buffer reagents used in the immunoprecipitation experiments were obtained from Wako Pure Chemicals, Ind. Biotin conjugated goat anti-rabbit IgG H&L (ab207995) and ATTO495-Biotin were from Abcam Co. and ATTO-TEC GmbH, respectively.
TCO-PEG3-MAL, MTZ-PEG4-MAL, mPEG-MTZ, MTZ-PEG4-sNHS, Sulfo-Cy3-MTZ, TCO-Amine and MTZ-PEG4-Amine were purchased from Click Chemistry Tools. Sulfo-Cy3-TCO was from AAT Bioquest, Inc. A product of Protein G conjugated magnetic beads (SureBeads Protein G) was obtained from Bio-Rad Laboratories. A high-performance size-exclusion chromatography column (Superdex 200 Increase 10/300 GL, bed dimensions: 10 × 300 mm, bed volume: approximately 24 ml) was purchased from GE healthcare. Other chemical reagents and devices of biochemical grade were as described in the previous paper [20]. Chemical structures were drawn using ACD/Chemsketch (Free ware) 2016.1.1. A densitometry analysis of the protein bands on an SDS-PAGE gel was performed using Image J [37].
In the following experiments, all protein sample concentration was conducted using an Amicon Ultra 15 [molecular-weight cut off (MWCO): 10 kDa] device by the centrifugation of 5000 G at 277 K. The size-exclusion chromatography fractionation by a disposable column in gravity-flow mode was performed using a PD-10 column (GE healthcare). High-performance size-exclusion chromatography was carried out using a Superdex 200 Increase 10/300 GL column under the conditions of 50 mM tris-hydrochloride containing 150 mM sodium chloride (pH 7.5) [50 mM Tris-HCl plus 150 mM NaCl (pH 7.5)] as the elution buffer and flow rate of 0.75 ml / min. In these conditions, the peak retention time of Ovalbumin (43 kDa), Aldolase (158 kDa) and Thyrogloblin (669 kDa) was 20.10 min, 17.73 min and 12.61 min, respectively. All sample solutions of the TCO- and MTZ-groups containing compounds were kept frozen at 253 K until use. Protein concentration was determined by a BCA protein assay kit using bovine serum albumin as the standard sample. SDS-PAGE analyses were performed using a 10–20% gradient gel and the protein bands were visualized by silver stain.
Preparation of TCO- and MTZ-derivatives of NFK3G1CG4-hFasLECD
The NFK3G1CG4-hFasLECD sample used for the preparation of either TCO- or MTZ-derivative was purified by a cation-exchange column chromatography (Hi-Trap SP, 5 ml) as described [19]. The protein concentration of the purified sample was determined to be 9.1 mg / ml. Freshly prepared twenty-fold molar excess amount each of TCO-PEG3-MAL and MTZ-PEG4-MAL in dehydrated dimethyl sulfoxide (dry DMSO) was used for the modification reactions to obtain the TCO- and MTZ-derivatives, respectively. Other details in experimental procedures were the same as described for the preparation of fluorescein 5-maleimide derivative in the previous paper [20], except for the substitution of the final purification step using the high-performance size-exclusion chromatography with the concentration step after the second size-exclusion chromatography in a gravity-flow mode. Typically, final recovery yields of the purified samples were 5.9 mg and 6.9 mg with respect to the TCO-derivative (hFasLECD-TCO) and the MTZ-derivatives (hFasLECD-MTZ) starting from 12 mg each of the purified NFK3G1CG4-hFasLECD samples, respectively.
Reactions of hFasLECD-TCO with mPEG-MTZ
Twenty μl (50 μg, 2.8 nmoles as the monomer subunit) each of hFasLECD-TCO (2.5 mg/ml) in 50 mM sodium acetate (pH 5.5) was mixed with 1.4 μl (0.5 M excess), 2.8 μl (1.0 M excess), 3.1 μl (1.1 M excess) or 4.1 μl (1.5, molar excess) of mPEG-MTZ (5 kDa) solution (5 mg / ml in deionized water). The reaction mixture was incubated for 1 h at 297 K, and then subjected to an SDS-PAGE analysis.
Preparation of sulfo-Cy3 conjugated NFK3G1CG4-hFasLECDs
For the conjugation with sulfo-Cy3-MTZ, 3.3 ml (5.5 mg, 0.30 μmole as the monomer subunit) of hFasLECD-TCO solution in 50 mM sodium acetate (pH 5.5) was mixed with 330 μl (0.41 μmole, a 1.4 M excess amount) of sulfo-Cy3-MTZ solution (1.1 μmoles / ml in deionized water). The reaction mixture was incubated for 1 h at 297 K. The same procedure was conducted using 3.3 ml (6.5 mg, 0.36 μmole as the monomer subunit) of hFasLECD-MTZ solution in 50 mM sodium acetate (pH 5.5) and 436 μl (0.48 μmole, a 1.3 M excess amount) of sulfo-Cy3-TCO solution (1.1 μmoles / ml in deionized water) for the conjugation of sulfo-Cy3-TCO. In either case, the reaction mixture after the incubation period was immediately resolved by two tandem steps of the chromatography in a gravity flow mode using 50 mM sodium acetate (pH 5.5) as the elution buffer. In the first resolving step, the reaction mixture sample was divided into two equivalent volume aliquots for a single application to the column, and one ml each fraction was collected into the reservoirs. The combined early four fractions eluted as pink, clear solutions were concentrated to approximately 2.0 ml. Then, the concentrate was subjected to the second resolving step for removing the remaining low molecular-weight contaminants completely. Finally, the sample was purified by the high performance size-exclusion chromatography. A 230 μl each aliquot of the sample was applied to the column in an individual run. The single main peak fractions showing the absorbance of 280 nm and 550 nm at the identical retention time were collected. The fractionated samples were combined and concentrated to a fluorescent deep pink, clear solution. The final recovery yield and the protein concentration in parenthesis were 2.8 mg (1.3 mg / ml) and 3.1 mg (0.92 mg / ml) with regard to Sulfo-Cy3-MT-hFasLECD and Sulfo-Cy3-TM-hFasLECD, respectively. The samples were kept frozen at 253 K in the dark until use, and then subjected to the SDS-PAGE analyses, the spectroscopic measurements and the experiments for the detection of complex formation with hFasRECD-Fc using the co-immunoprecipitation and the high-performance size-exclusion chromatography analyses.
Preparation of avidin-hFasLECD
Avidin-hFasLECD was synthesized by the conjugation of Avidin-MTZ with hFasLECD-TCO. Avidin-MTZ (Fig. 1b) was prepared by the reaction of a commercially available biochemical grade avidin from chicken egg-white with eightfold molar excess amount of MTZ-PEG4-sNHS as follows. Ten mg of avidin was dissolved in 2.0 ml of 0.1 M sodium hydrogen carbonate (pH 8.3), then 75 μl of MTZ-PEG4-sNHS solution (2 mg in 200 μl deionized water) prepared immediately before the reaction was added. The reaction mixture was incubated for 4 h at 301 K. After that, the reaction mixture was quenched with 140 μl of 1 M Tris HCl (pH 7.5) and further incubated for 15 min. The quenched sample was resolved by the size-exclusion chromatography in a gravity-flow mode using 50 mM Tris-HCl plus 150 mM NaCl (pH 7.5) as the elution buffer. The same resolution step was repeated again to remove the low molecular-weight contaminants containing MTZ group completely. The recovered sample was concentrated to 2.4 ml (4.3 mg/ml) of a pale pink, clear solution, and used as the sample for the following conjugation reactions.
Initial attempts of the conjugation reaction between Avidin-MTZ and hFasLECD-TCO were performed by mixing 10 μl, 20 μl or 30 μl of hFasLECD-TCO solution [2.52 mg/ml in 50 mM sodium acetate (pH 5.5)] with a series (1.0, 1.2, 1.5 or 3.0 M excess amount) of Avidin-MTZ solutions [4.3 mg/ml in 50 mM Tris-HCl plus 150 mM NaCl (pH 7.5)], and incubated for 1 h at 301 K. Each reaction mixture was diluted to 200 μl with 50 mM Tris-HCl plus 150 mM NaCl (pH 7.5) buffer¸ and then subjected to an analysis using the high performance size-exclusion chromatography. A large scale conjugation reaction under the condition of 1.5 fold excess molar amount of Avidin-MTZ relative to hFasLECD-TCO was conducted by mixing 1.1 ml (2.7 mg, 70 nmoles) of Avidin-MTZ solution with 1.0 ml (2.5 mg, 46 nmoles) of hFasLECD-TCO solution. The reaction mixture was incubated for 1 h at 299 K, and then quenched with 23 μl of 30 mM TCO-Amine solution (3.9 mg in 0.5 ml of deionized water) by incubating for further 1 h. The final colorless, clear reaction mixture after the quenching reaction was applied to a single step of the size-exclusion chromatography in a gravity-flow mode to remove the low molecular-weight contaminants, and then 230 μl aliquots of the recovered sample were resolved by the high performance size-exclusion column chromatography to obtain single peak fractions. All isolated fractions were combined together and concentrated to 1.4 ml for the analyses in the following experiments (recovery yield, 1.5 mg).
Preparation of rFab’-hFasLECDs
rFab’-hFasLECDs were synthesized by the conjugation of rFab’-MTZ with hFasLECD-TCO. The rFab’ domain was obtained essentially according to the procedures described in the previous papers [38, 39]. Thirty five mg of the commercially available Protein A purified normal rabbit IgG whole molecule in 3.5 ml of 0.1 M sodium acetate containing 0.1 M sodium chloride buffer (pH 4.5) was digested with 1.6 mg of Pepsin from porcine stomach by incubating for 20 h at 310 K (Additional file 3a). The sample after the digestion was subjected to exchange the buffer with 50 mM Tris-HCl plus 150 mM NaCl (pH 7.5) by the size-exclusion column chromatography in a gravity-flow mode. Then, 230 μl aliquots of the sample were further fractionated by the high performance size-exclusion chromatography using the same buffer (Additional file 3b, left panel). The main peak fractions containing rF(ab’)2 were collected and combined to total sample volume of 32.0 ml. The sample was concentrated to 3.6 ml (5.4 mg/ml). To a half volume of this sample solution containing 9.8 mg (0.21 μmole) of rF(ab’)2, 48 μl of 0.5 M ethylenediaminetetraacetic acid sodium salt (EDTA-Na) (pH 8.0) and 240 μl of freshly prepared 100 mM 2-aminoethantiol hydrochloride solution in 50 mM Tris-HCl containing 10 mM EDTA-Na (pH 7.5) were added and incubated for 30 min at 310 K, for the conversion of rF(ab’)2 to rFab’. Then, the reaction mixture was immediately subjected to a size-exclusion chromatography column pre-equilibrated with 25 mM sodium phosphate containing 0.1 M sodium chloride and 5 mM EDTA-Na (pH 6.4) for buffer-exchange. The sample containing rFab’ was diluted to 9.7 ml with the same buffer, and freshly prepared MTZ-PEG4-MAL solution [10 mg (19 μmoles) in 0.97 ml of dry DMSO] was added. The reaction mixture was incubated for 3 h at 297 K, and then quenched with 22 μl of 1 M L-cysteine hydrochloride solution in deionized water by incubating further 1 h. The quenched reaction mixture was concentrated to 2.0 ml, and further subjected to the two tandem size-exclusion chromatography in a gravity-flow mode to remove the MTZ-group containing low molecular-weight contaminants completely. After that, the high-performance size-exclusion chromatography resolutions of 230 μl aliquots were performed to obtain the main peak fractions of rFab’-MTZ sample (Additional file 3b, right panel). The collected samples were combined and concentrated to 3.0 ml of pale pink, clear solution (recovery yield 6.9 mg, 2.3 mg/ml).
Initial attempts of the conjugation reaction between rFab’-MTZ and hFasLECD-TCO were performed by mixing 10 μl each of hFasLECD-TCO solution [2.5 mg / ml in 50 mM sodium acetate (pH 5.5)] with a series (1.0, 2.0, 3.0 or 5.0 M excess amount) of rFab’-MTZ solutions [2.3 mg / ml in 50 mM Tris-HCl plus 150 mM NaCl (pH 7.5)] and incubated for 1 h at 298 K. Each reaction mixture was diluted to 200 μl with 50 mM Tris-HCl plus 150 mM NaCl (pH 7.5) buffer for subjecting to an analysis by the high-performance size-exclusion column chromatography. Large scale conjugation reactions under the condition of 1.0 M excess and 5.0 M excess amounts of rFab’-MTZ relative to hFasLECD were conducted by mixing 1.2 ml (2.7 mg, 58 nmoles) of rFab’-MTZ solution with 1.3 ml (3.2 mg, 60 nmoles) of hFasLECD-TCO solution, and 1.5 ml (3.4 mg, 72 nmoles) of rFab’-MTZ solution with 0.31 ml (0.78 mg, 14 nmoles) of hFasLECD-TCO solution, respectively. Both reaction mixtures were incubated for 1 h at 298 K, and then quenched by incubating for further 1 h with 19 μl (in the 1.0 M excess amount reaction) and 4.8 μl (in the 5.0 M excess amount reaction) of 30 mM MTZ-PEG4-Amine solutions (5.0 mg in 0.42 ml of deionized water), respectively. The final pale pink, clear solutions were subjected to the size-exclusion chromatography in a gravity mode. Then, 230 μl aliquots were resolved using the high-performance size-exclusion column chromatography to obtain the fractionated samples. The isolated sample fractions combined together were concentrated to 1.0 ml (0.57 mg) and 0.88 ml (0.13 mg) with regard to the reaction using 1.0 M excess amount of rFab’-MTZ and that using the 5.0 M excess amount of rFab’-MTZ, respectively.
Preparation of the complex between avidin-hFasLECD and ATTO495-biotin
1.2 ml (1.2 mg) of the isolated avidin-hFasLECD conjugate was mixed with 40 μl of ATTO495-Biotin solution (1 mg in 100 μl of Dry DMSO) and incubated for 2 h on ice. The mixture was resolved by the two tandem steps of chromatography in a gravity-flow mode in order to completely remove the free ATTO495-Biotin. The sample recovered in the second resolving step (0.84 mg, 240 μg / ml) was subjected to the experiment for detection of the complex.
Spectroscopic measurements and estimation of conjugation number of sulfo-Cy3
UV-Vis absorption spectra in the range from 250 nm to 650 nm, a couple of independent measurements of absorption values at 280 nm and 552 nm used for the calculation of an estimated conjugation number of sulfo-Cy3 groups to hFasLECD and fluorescent spectra measurement under the condition of the excitation wavelength at 552 nm were performed as described in the previous paper [20]. All measurements were conducted under the sample concentrations of 125 μg / ml. In the calculation of the estimated conjugation number, the correction factor of sulfo-Cy3 group at 280 nm was set to 0.05, and the molar extinction coefficient of sulfo-Cy3 group was assumed as 150,000 [40]. The molar extinction coefficient of NFK3G1CG4-hFasLECD was obtained as 29,005 using the Prot Param tool on the EXPAsy Server [41].
Detection of the complex formation
Detection of the specific binding activity of the isolated conjugates, i.e. sulfo-Cy3-hFasLECDs, Avidin-hFasLECD and rFab’-hFasLECDs, and the components of the conjugates, i.e. hFasLECD-TCO, Avidin-MTZ and rFab’-MTZ, (5.5 μg each) toward either the hFasRECD-Fc sample (8.8 μg) or biotin conjugated goat anti-rabbit IgG H&L (14.0 μg) were conducted using a Protein G conjugated magnetic beads (1.0 mg) as the precipitating agent by the receptor- or the antibody-mediated co-immunoprecipitation in 1.0 ml of 50 mM Tris-HCl plus 150 mM NaCl buffer (pH 7.5) containing 1% Nonidet P40 and 0.5% sodium deoxycholate, as described in the previous paper [25]. Another experiment for the detection of the complex formation between sulfo-Cy3-hFasLECDs and hFasRECD-Fc was also performed by the high-performance size-exclusion chromatography using the mixture solutions composed of sulfo-Cy3-hFasLECDs (7.5 μg each) and hFasRECD-Fc (19.4 μg) in 230 μl solution as described in the previous paper [20]. The UV-Vis spectra of the isolated complex sample of Avidin-hFasLECD conjugate with ATTO495-Biotin and the Avidin-hFasLECD conjugate alone sample were compared at the concentration of 240 μg / ml in 50 mM Tris-HCl plus 150 mM NaCl (pH 7.5). A solution of free ATTO495-Biotin showing the absorbance value at 495 nm (0.29) similar to that of the isolated complex sample (0.26) was also subjected to measurement for comparison of the absorption peak profiles.
