Rf 550

The culture supernatant was obtained by centrifugation and 10 μl of supernatant was mixed with an equal volume of SDS sample buffer (Bio-Rad, Hercules, CA, USA) without reducing agent. Proteins were separated by 10%–20% gradient polyacrylamide gel electrophoresis (PAGE) as described by Laemmli [47 (link)] under non-reducing conditions, and the gels were stained with SYPRO Orange (Life Technologies, Carlsbad, CA, USA). Estimation of Fab production yield was done by quantifying the intensity of protein bands in gels using Multi Gauge software (Fujifilm, Tokyo, Japan). A standard curve was generated using a standard protein solution of known concentration run in the same gel. For determining the N-terminal amino acid sequence, proteins were transferred to a polyvinylidene difluoride (PVDF) membrane by electroblotting after separation by sodium dodecyl sulfate (SDS)-PAGE, and the protein bands were directly applied to a gas-phase protein sequencer (model PSQ; Shimadzu, Kyoto, Japan) equipped with an in-line amino acid analyzer (model RF-550; Shimadzu), as described previously [48 (link)]. Western blotting analysis was performed with alkaline phosphatase (AP) conjugated anti-human IgG(H+L) antibody (Rockland Immunochemicals, Gilbertsville, PA, USA) and an AP conjugate substrate kit (Bio-Rad, Hercules, CA, USA).

Basic procedures for the measurement of content of three chlorophyll precursors, protoporphyrin IX, Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethylester were followed by an HPLC-based procedure as described previously [38 (link)]. Extraction of chlorophyll precursors from seedlings was carried out using the same procedure as for chlorophyll extraction described above. The acetone extracts were applied to a HPLC system (LC10A VP system, Shimadzu) equipped with a reverse phase column (Symmetry C8, 150 mm × 4.6 mm, 3.5μm particle size, Waters, http://www.waters.com/) with a guard column (C8, 4.0 mm × 3.0 mm, Phenomenex, http://www.phenomenex.com/). Chlorophyll precursors were eluted by an anomalous gradient from solvent A (methanol:acetonitrile: 0.25 M pyridine = 50:25:25) to solvent B (methanol:acetonitrile:acetone = 20:60:20). Eluents were monitored with a fluorescence detector (RF-550, Shimadzu). For protoporphyrin IX detection, the excitation wavelength was set at 400 nm and fluorescence emission was detected at 634 nm, while for detections of Mg-protoporphyrin IX and Mg-protoporphyrin IX monomethylester, the excitation wavelength was set at 417 nm and fluorescence emission was detected at 600 nm. Content of these precursors was estimated through calibration curves determined from standard substances (Frontier Scientific, http://www.frontiersci.com/).