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.