(A) α-TM was tested as an acceptor for the polymerization reaction, where co-expressed ChSy-1 and ChPF, CSS3 (ChSy-2) and ChPF, or CSS3 (ChSy-2) and ChSy-1 were used as an enzyme source as described in the Experimental section. [³H]GalNAc-labelled products were first isolated by gel-filtration, subjected to reductive β-elimination using NaBH₄/NaOH, and then rechromatographed on a Superdex 200 column (1.0 cm×30 cm) as shown in this Figure. Effluent fractions (0.4 ml each) were analysed for radioactivity. The broken line indicates commercial chondroitin chains (a chemically desulfated derivative of whale cartilage chondroitin sulfate A). ■, products obtained using co-expressed ChSy-1 and ChPF; ●, products obtained using co-expressed CSS3 (ChSy-2) and ChPF; ▲, products obtained using co-expressed CSS3 (ChSy-2) and ChSy-1; ○, products obtained using a mixture of separately expressed soluble forms of CSS3 (ChSy-2) and ChPF. The inset shows the calibration curve, showing a linear relation between the log M_r and the elution volume, which was generated using the data obtained with size-defined commercial polysaccharides; dextran (average M_r: 200000, 65500, 37500, and 18100; all from Sigma). (B) The [³H]GalNAc-labelled products were first isolated by gel-filtration and subjected to reductive β-elimination using NaBH₄/NaOH. The ³H-labelled products were digested with chondroitinase ABC and chromatographed on a Superdex peptide column (1.0 cm×30 cm). Effluent fractions (0.4 ml each) were analysed for radioactivity. The arrowhead indicates the elution position of the authentic disaccharide, GlcAβ1–3GalNAc. ●, products obtained using co-expressed CSS3 (ChSy-2) and ChPF; ▲, products obtained using co-expressed CSS3 (ChSy-2) and ChSy-1.

Culture medium from cells co-expressing ChSy-2–ProA and ChPF–His, ChSy-1–His, or EXT-1–His, co-expressing ChSy-1–ProA and ChPF–His, or co-expressing EXT-2–ProA and EXT-1–His was purified with Ni²⁺-NTA agarose and subjected to SDS/PAGE. Separated proteins were transferred to PVDF membrane by Western blotting, allowed to react with IgG as a primary antibody, and visualized using the ECL® advance kit. Lane 1, ChSy-2–ProA and ChSy-1–His; lane 2, ChSy-2–ProA and ChPF–His; lane 3, ChSy-1–ProA and ChPF–His; lane 4, ChSy-2–ProA and EXT-1–His; lane 5, EXT-2–ProA and EXT-1–His.

Northern blots with RNA from various human tissues were hybridized with a probe for CSS3 (ChSy-2), ChSy-1, ChPF or β-actin as described in the Experimental section. Lane 1, brain; lane 2, heart; lane 3, skeletal muscle; lane 4, colon; lane 5, thymus; lane 6, spleen; lane 7, kidney; lane 8, liver; lane 9, small intestine; lane 10, placenta; lane 11, lung; lane 12, peripheral blood leucocytes.