Production of the extracellular domain of Siglec-9 using and an E.coli expression system and generation of anti-Siglec-9 antibodies

694 Sialic acid-binding immunoglobulin-like lectins (Siglecs) are a subset of the immunogluobulin superfamily of cell surface receptors that mediate protein-carbohydrate interactions through their ability to bind sialic acid moieties found on glycolipids and glycoproteins. Siglecs are mainly expressed on white blood cells and play a critical role in cell-cell interactions and signaling functions in the hematopoietic, immune, and nervous systems. The human Siglec family is composed of 11 genes. In addition to well-characterized Siglecs such as the myeloid receptor (Siglec-3/CD33), seven additional Siglecs (Siglec 5-11) with high sequence similarity to Siglec-3 and collectively referred to as Siglec-3-related genes have been identified in recent years. Anti-CD33/Siglec-3 antibodies have recently been used for diagnosis of acute myeloid leukemia (AML). The development of Siglec-3 antibody-targeted chemotherapy, as well as emergence of anti-Siglec-5 antibodies as a potential marker and therapeutic reagent for AML has prompted the characterization of other newly identified Siglecs. Siglec-9 is one of the newly identified members of the Siglec gene family. Similar to other Siglec-3-related genes, Siglec-9 is located on chromosome 19q 13.3-4 in close proximity to the kallikrein gene locus. Siglec-9 is composed of an N-terminal V-set Ig domain that mediates sialic acid binding, two C2-set Ig domains, a transmembrane region and a cytoplasmic tail that harbors two immune receptor tyrosine-based inhibitory motifs (ITIMs). Here we report the production of the extracellular domain of Siglec-9 using an E.coli protein expression system. Total bone marrow mRNA was reverse-transcribed to cDNA. Polymerase chain reaction using Siglec-9 specific primers was conducted. The PCR product was cloned into pET-200 TOPO plasmid vector and the construct was used to transform E.coli strain BL21(DE3) for protein production. Following identification by mass spectroscopy, recombinant Siglec-9 containing an N-terminal polyhistidine (6xHis) tag was purified to homogeneity using metal affinity chromatography and was used as immunogen for antibody production in rabbit and mice. Anti-Siglec-9 polyclonal and monoclonal antibodies were produced and used in flow-cytometric analysis of lymph nodes obtained from patients with hematological malignancies. 7 out of 8 patients with B-cell lymphoma were positive. We conclude that Siglec-9 may be a new surface marker of patients with B-cell lymphoma.