Functional Expression and Characterization of the Recombinant N-Acetyl-Glucosamine/N-Acetyl-Galactosamine-Specific Marine Algal Lectin BPL3

Abstract

Lectins, characterized by their carbohydrate-binding ability, have extensive practical applications. However, their industrial use is limited due to impurity. Thus, quality-controlled production of recombinant lectin is necessary. In this study, the algal lectin BPL3 (Bryopsis plumosa lectin 3) was successfully produced using a bacterial expression system, BL21(DE3), with an artificial repeated structure (dimeric construct). Recombinant dimeric BPL3 (rD2BPL3) was confirmed by LC-MS/MS spectrometry. Expression efficiency was greater for the construct with the repeat structure (rD2BPL3) than the monomeric form (rD1BPL3). Optimal conditions for expression were 1 mM IPTG at 20 °C. Recombinant lectin was purified under denaturing conditions and refolded by the flash dilution method. Recombinant BPL3 was solubilized in 1× PBS containing 2 M urea. rD2BPL3 showed strong hemagglutination activity using human erythrocyte. rD2BPL3 had a similar sugar specificity to that of the native protein, i.e., to N-acetyl-glucosamine (GlcNAc) and N-acetyl-galactosamine (GalNAc). Glycan array results showed that recombinant BPL3 and native BPL3 exhibited different binding properties. Both showed weak binding activity to α-Man-Sp. Native BPL3 showed strong binding specificity to the alpha conformation of amino sugars, and rD2BPL3 had binding activity to the beta conformation. The process developed in this study was suitable for the quality-controlled large-scale production of recombinant lectins.

Characterization of a Novel Mannose-Binding Lectin with Antiviral Activities from Red Alga, Grateloupia chiangii

“…This may contribute to the stability of lectin's tertiary structure and may be a substitute for the advantages conferred through multimeric structures [18,34]. The tandem repeat structure of GCL was considered to be preferable for our purposes, as this is shared with other red algal lectins and may contribute to an increase in hemagglutination activity [18,23,34]. Furthermore, the isolated GCL amino acid sequence interestingly represented high similarity with bacterial lectins as shown in Supplementary Figure S4.…”

“…Peptide mapping was performed according to previous methods [23]. Protein bands obtained from SDS-PAGE were excised, in-gel digested with trypsin, and cleaned with Zip-Tip (Millipore, Billerica, MA, USA).…”

“…A glycan microarray analysis was performed by Ebiogen (Seoul, Korea) according to previous methods [23]. The Glycan Array kit was purchased from RayBioTech (Norcross, GA, USA).…”

“…An array containing 300 synthetic glycans printed in quadruplicate on a glass slide was used. Label-based detection was performed according to the manufacturer's protocols and previous methods [23]. Biotinylated recombinant lectins and native lectins at 50 µg/mL were added to the array wells and incubated for >3 h with gentle rocking.…”

“…Several red algal lectins possess a unique repeated primary structure [18] and the contribution of this tandem repeat structure to lectin activity was elucidated by recombinant technology [18,23]. These studies revealed that the primary structure of red algal lectin contributes to the production of lectin by recombinant proteins.…”