Functional Expression and Characterization of the Recombinant N-acetyl-

22 23 Lectins, characterized by their carbohydrate-binding ability, have an extensive practical application. 24 However, their industrial use is limited by low yields, and few active recombinant lectins have been 25 reported. In this study, the algal lectin BPL-3 (Bryopsis plumosa lectin 3) was successfully produced 26 using a bacterial expression system, BL21(DE3), with an artificial repeated structure (dimeric 27 construct). Recombinant dimeric BPL3 (rD2BPL3) was confirmed by LC-MS/MS spectrometry. 28 Expression efficiency was greater for the construct with the repeat structure (rD2BPL3) than the 29 monomeric form (rD1BPL3). Optimal conditions for expression were 1 mM IPTG at 20 °C. 30 Recombinant lectin was purified under denaturing conditions and refolded by the flash dilution 31 method. Recombinant BPL3 was solubilized in 1× PBS containing 2 M urea. rD2BPL3 showed strong 32 hemagglutination activity using human erythrocytes, similar to that of native BPL3. rD2BPL3 had a 33 similar sugar specificity to that of the native protein, i.e., to N-acetyl-glucosamine (GlcNAc) and N34 acetyl-galactosamine (GalNAc). Glycan array results showed that recombinant BPL3 and native 35 BPL3 exhibited different binding properties. Both showed weak binding activity to α-Man-Sp. Native 36 BPL3 showed strong binding specificity to the alpha conformation of amino sugars, and rD2BPL3 37 had binding activity to the beta conformation. The process developed in this study was suitable for the 38 quality-controlled production of high amounts of soluble recombinant lectins. 39 40