Fibronectin inhibits osteoclastogenesis while enhancing osteoclast activity via nitric oxide and interleukin‐1β‐mediated signaling pathways

Osteoclasts are bone‐resorbing cells formed by fusion of mononuclear precursors. The matrix proteins, fibronectin (FN), vitronectin (VN), and osteopontin (OPN) are implicated in joint destruction and interact with osteoclasts mainly through integrins. To assess the effects of these matrix proteins on osteoclast formation and activity, we used RAW 264.7 (RAW) cells and mouse splenocytes differentiated into osteoclasts on tissue culture polystyrene (TCP) or osteologic? slides pre‐coated with 0.01–20 µg/ml FN, VN, and OPN. At 96 h, osteoclast number and multinucleation were decreased on VN and FN compared to OPN and TCP in both RAW and splenocytes cell cultures. When early differentiation was assessed, VN but not FN decreased cytoplasmic tartrate‐resistant acid phosphatase activity and pre‐osteoclast number at 48 h. OPN had the opposite effect to FN on osteoclast formation. When RAW cells were differentiated on OPN and treated by FN and OPN, osteoclast number only in the FN treated group was 40–60% lower than the control, while the total number of nuclei was unchanged, suggesting that FN delays osteoclast fusion. In contrast to its inhibitory effect on osteoclastogenesis, FN increased resorption by increasing both osteoclast activity and the percentage of resorbing osteoclasts. This was accompanied by an increase in nitric oxide (NO) levels and interleukin‐1β (IL‐1β). IL‐1β production was inhibited using the NO‐synthase inhibitor only on FN indicating a FN‐specific cross‐talk between NO and IL‐1β signaling pathways. We conclude that FN upregulates osteoclast activity despite inhibiting osteoclast formation and that these effects involve NO and IL‐1β signaling. J. Cell. Biochem. 111: 1020–1034, 2010. © 2010 Wiley‐Liss, Inc.     

Dual‐specificity tyrosine phosphorylation‐regulated kinase 2 regulates osteoclast fusion in a cell heterotypic manner

Monocyte fusion into osteoclasts, bone resorbing cells, plays a key role in bone remodeling and homeostasis; therefore, aberrant cell fusion may be involved in a variety of debilitating bone diseases. Research in the last decade has led to the discovery of genes that regulate osteoclast fusion, but the basic molecular and cellular regulatory mechanisms underlying the fusion process are not completely understood. Here, we reveal a role for Dyrk2 in osteoclast fusion. We demonstrate that Dyrk2 down regulation promotes osteoclast fusion, whereas its overexpression inhibits fusion. Moreover, Dyrk2 also promotes the fusion of foreign‐body giant cells, indicating that Dyrk2 plays a more general role in cell fusion. In an earlier study, we showed that fusion is a cell heterotypic process initiated by fusion‐founder cells that fuse to fusion‐follower cells, the latter of which are unable to initiate fusion. Here, we show that Dyrk2 limits the expansion of multinucleated founder cells through the suppression of the fusion competency of follower cells.     

TNF‐α has both stimulatory and inhibitory effects on mouse monocyte‐derived osteoclastogenesis

Phenotypically different osteoclasts may be generated from different subsets of precursors. To what extent the formation of these osteoclasts is influenced or mediated by the inflammatory cytokine TNF‐α, is unknown and was investigated in this study. The osteoclast precursors early blasts (CD31^hiLy‐6C^?), myeloid blasts (CD31⁺Ly‐6C⁺), and monocytes (CD31^?Ly‐6C^hi) were sorted from mouse bone marrow using flow cytometry and cultured with M‐CSF and RANKL, with or without TNF‐α. Surprisingly, TNF‐α prevented the differentiation of TRAcP⁺ osteoclasts generated from monocytes on plastic; an effect not seen with early blasts and myeloid blasts. This inhibitory effect could not be prevented by other cytokines such as IL‐1β or IL‐6. When monocytes were pre‐cultured with M‐CSF and RANKL followed by exposure to TNF‐α, a stimulatory effect was found. TNF‐α also stimulated monocytes’ osteoclastogenesis when the cells were seeded on bone. Gene expression analysis showed that when TNF‐α was added to monocytes cultured on plastic, RANK, NFATc1, and TRAcP were significantly down‐regulated while TNF‐αR1 and TNF‐αR2 were up‐regulated. FACS analysis showed a decreased uptake of fluorescently labeled RANKL in monocyte cultures in the presence of TNF‐α, indicating an altered ratio of bound‐RANK/unbound‐RANK. Our findings suggest a diverse role of TNF‐α on monocytes’ osteoclastogenesis: it affects the RANK‐signaling pathway therefore inhibits osteoclastogenesis when added at the onset of monocyte culturing. This can be prevented when monocytes were pre‐cultured with M‐CSF and RANKL, which ensures the binding of RANKL to RANK. This could be a mechanism to prevent unfavorable monocyte‐derived osteoclast formation away from the bone.

     

Osteoclast formation is strongly reduced both in vivo and in vitro in the absence of CD47/SIRPalpha-interaction

Physical interaction between the cell surface receptors CD47 and signal regulatory protein alpha (SIRPalpha) was reported to regulate cell migration, phagocytosis, cytokine production, and macrophage fusion. However, it is unclear if the CD47/SIRPalpha-interaction can also regulate macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-kappaB ligand (RANKL)-stimulated formation of osteoclasts. Here, we show that functional blocking antibodies to either CD47 or SIRPalpha strongly reduced formation of multinucleated tartrate-resistant acid phosphatase (TRAP)+ osteoclasts in cultures of murine hematopoietic cells, stimulated in vitro by M-CSF and RANKL. In addition, the numbers of osteoclasts formed in M-CSF/RANKL-stimulated bone marrow macrophage cultures from CD47-/- mice were strongly reduced, and bones of CD47-/- mice exhibited significantly reduced osteoclast numbers, as compared with wild-type controls. We conclude that the CD47/SIRPalpha interaction is important for M-CSF/RANKL-stimulated osteoclast formation both in vivo and in vitro, and that absence of CD47 results in decreased numbers of osteoclasts in CD47-/- mice.     

Effect of CD44 deficiency on in vitro and in vivo osteoclast formation

In vitro studies have shown that CD44 is involved in the fusion process of osteoclast precursor cells. Yet, in vivo studies do not support this, since an osteopetrotic phenotype has not been described for CD44 knock-out (CD44 k.o.) mice. This discrepancy may suggest that the role of CD44 in fusion may depend on the microenvironment of osteoclast formation. We investigated osteoclast formation of CD44 k.o. and wild-type mice under three conditions: in vitro, both on plastic and on bone and in vivo by analyzing osteoclast number, and size in long bones from wild-type and CD44 k.o. mice. Bone marrow cells from wild-type and CD44 k.o. mice were analyzed for their capacity to form osteoclasts on plastic and on bone in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). On plastic, the number of multinucleated tartrate resistant acid phosphatase (TRAP) positive cells in CD44 k.o. cultures was twofold higher than in wild-type cultures. On bone, however, equal numbers of osteoclasts were formed. Interestingly, the total number of osteoclasts formed on bone proved to be higher than on plastic for both genotypes, strongly suggesting that osteoclastogenesis was stimulated by the bone surface, and that CD44 is not required for osteoclast formation on bone. Functional analyses showed that bone resorption was similar for both genotypes. We further studied the osteoclastogenic potential of wild-type bone marrow cells in the presence of CD44 blocking antibodies. Osteoclastogenesis was not affected by these antibodies, a further indication that CD44 is not required for the formation of multinucleated cells. Finally, we analyzed the in vivo formation of osteoclasts by analyzing long bones from wild-type and CD44 k.o. mice. Morphometric analysis revealed no difference in osteoclast number, nor in number of nuclei per osteoclasts or in osteoclast size. Our in vitro experiments on plastic showed an enhanced formation of osteoclasts in the absence of CD44, thus suggesting that CD44 has an inhibitory effect on osteoclastogenesis. However, when osteoclasts were generated on bone, no differences in number of multinucleated cells nor in bone resorption were seen. These observations are in agreement with in vivo osteoclast characteristics, where no differences between wild-type and CD44 k.o. bones were encountered. Therefore, the modulating role of CD44 in osteoclast formation appears to depend on the microenvironment.     

Prostate cancer promotes CD11b positive cells to differentiate into osteoclasts

Bone is the preferred site of prostate cancer metastasis, contributing to the morbidity and mortality of this disease. A key step in the successful establishment of prostate cancer bone metastases is activation of osteoclasts with subsequent bone resorption causing the release of several growth factors from the bone matrix. CD11b+ cells in bone marrow are enriched for osteoclast precursors. Conditioned media from prostate cancer PC‐3 cells induces CD11b+ cells from human peripheral blood to differentiate into functional osteoclasts with subsequent bone resorption. Analysis of PC‐3 conditioned media revealed high amounts of IL‐6 and IL‐8. CD11b+ cells were cultured with M‐CSF and RANKL, IL‐6, IL‐8, and CCL2, alone or in combination. All of these conditions induced osteoclast fusion, but cells cultured with M‐CSF, IL‐6, IL‐8, and CCL2 were capable of limited bone resorption. Co‐incubation with IL‐6 and IL‐8 and the RANK inhibitor, RANK‐Fc, failed to inhibit osteoclast fusion and bone resorption, suggesting a potential RANKL‐independent mechanism of functional osteoclast formation. This study demonstrates that functional osteoclasts can be derived from CD11b+ cells derived from human PBMCs. Prostate cancer cells secrete factors, including IL‐6 and IL‐8, that play an important role in osteoclast fusion by a RANKL‐independent mechanism. J. Cell. Biochem. 106: 563–569, 2009. © 2009 Wiley‐Liss, Inc.     

Direct cell–cell contact between periodontal ligament fibroblasts and osteoclast precursors synergistically increases the expression of genes related to osteoclastogenesis

The formation of bone resorbing osteoclasts in vivo is orchestrated by cells of the osteoblast lineage such as periodontal ligament fibroblasts that provide the proper signals to osteoclast precursors. Although the requirement of cell–cell interactions is widely acknowledged, it is unknown whether these interactions influence the expression of genes required for osteoclastogenesis and the ultimate formation of osteoclasts. In the present study we investigated the effect of cell–cell interaction on the mRNA expression of adhesion molecules and molecules involved in osteoclast formation in cultures of peripheral blood mononuclear cells (PBMCs) and human primary periodontal ligament fibroblasts, both as solitary cultures and in co‐culture. We further analyzed the formation of multinucleated, tartrate resistant acid phosphatase (TRACP) positive cells and assessed their bone resorbing abilities. Interestingly, gene expression of intercellular adhesion molecule‐1 (ICAM‐1) and of osteoclastogenesis‐related genes (RANKL, RANK, TNF‐α, and IL‐1β) was highly up‐regulated in the co‐cultures compared to mono‐cultures and the 5–10‐fold up‐regulation reflected a synergistic increase due to direct cell–cell interaction. This induction strongly overpowered the effects of known osteoclastogenesis inducers 1,25(OH)₂VitD₃ and dexamethasone. In case of indirect cell–cell contact mRNA expression was not altered, indicating that heterotypic adhesion is required for the increase in gene expression. In addition, the number of osteoclast‐like cells that were formed in co‐culture with periodontal ligament fibroblasts was significantly augmented compared to mono‐cultures. Our data indicate that cell–cell adhesion between osteoclast precursors and periodontal ligament fibroblasts significantly modulates the cellular response which favors the expression of osteoclast differentiation genes and the ultimate formation of osteoclasts. J. Cell. Physiol. 222: 565–573, 2010. © 2009 Wiley‐Liss, Inc.     

Syncytin-1 in differentiating human myoblasts: relationship to caveolin-3 and myogenin

Myoblasts fuse to form myotubes, which mature into skeletal muscle fibres. Recent studies indicate that an endogenous retroviral fusion gene, syncytin-1, is important for myoblast fusions in man. We have now expanded these data by examining the immunolocalization of syncytin in human myoblasts induced to fuse. Additionally, we have compared the localization of syncytin with the localization of caveolin-3 and of myogenin, which are also involved in myoblast fusion and maturation. Syncytin was localized to areas of the cell membrane and to filopodial structures connecting myoblasts to each other and to myotubes. Weaker staining was present over intracellular vesicles and tubules. Caveolin-3 was detected in the sarcolemma and in vesicles and tubules in a subset of myoblasts and myotubes. The strongest staining occurred in multinucleated myotubes. Wide-field fluorescence microscopy indicated a partial colocalization of syncytin and caveolin-3 in a subset of myoblasts. Super-resolution microscopy showed such colocalization to occur in the sarcolemma. Myogenin was restricted to nuclei of myoblasts and myotubes and the strongest staining occurred in multinucleated myotubes. Syncytin staining was observed in both myogenin-positive and myogenin-negative cells. Antisense treatment downmodulated syncytin-1 expression and inhibited myoblast cell fusions. Importantly, syncytin-1 antisense significantly decreased the frequency of multinucleated myotubes demonstrating that the treatment inhibited secondary myoblast fusions. Thus, syncytin is involved in human myoblast fusions and is localized in areas of contact between fusing cells. Moreover, syncytin and caveolin-3 might interact at the level of the sarcolemma.     

Comparison of morphological effects of PGE2 and TGFβ on osteoclastogenesis induced by RANKL in mouse bone marrow cell cultures

RANKL, in the presence of M-CSF, induces the development and fusion of TRAP+ osteoclasts in mouse bone marrow cultures at 3–5 days. Early during culture (day 3), most cells are small (up to six nuclei). At lower cell densities, these osteoclasts exhibit a rounded morphology with cytoplasm extending around the cells but, at higher densities, this changes to a stellate morphology with the cytoplasm being retracted around the nuclei with numerous localised cytoplasmic extensions. Under optimal conditions, osteoclast fusion results in conglomerates of many cells, which become large cytoplasmic masses on day 4. PGE2 and TGFβ have both been shown to increase osteoclast development in this model and their effects on the morphology of osteoclasts during fusion and differentiation have been compared under all these conditions. PGE2 or TGFβ increase osteoclast numbers and size and also the number of nuclei, indicating increased osteoclast development and fusion. TGFβ increases the size of rounded osteoclasts (with respect to the number of nuclei) more than PGE2, suggesting that TGFβ increases cytoplasmic extension. TGFβ increases the size and number of nuclei in stellate cells but particularly increases the number and length of the cytoplasmic extensions, in contrast to PGE2. Fusion of these extensions with other osteoclasts results in large networks of interconnected cells. On day 4, spreading cells develop but these are still interconnected by cytoplasmic links, a phenomenon not seen in control wells or after treatment with PGE2. TGFβ is more effective than PGE2 in increasing fusion in the formation of cell conglomerates and cytoplasmic masses. PGE2 decreases overall cell density resulting in additional indirect effects on osteoclast numbers and morphology. However, PGE2 particularly promotes the formation of large mature spreading osteoclasts later during culture.     

Imperatorin promotes osteogenesis and suppresses osteoclast by activating AKT/GSK3 β/β‐catenin pathways

Osteoporosis is caused by disturbance in the dynamic balance of bone remodelling, a physiological process, vital for maintenance of healthy bone tissue in adult humans. In this process, a new bone is formed by osteoblasts and the pre‐existing bone matrix is resorbed by osteoclasts. Imperatorin, a widely available and inexpensive plant extract with antioxidative and apoptotic effects, is reported to treat osteoporosis. However, the underlying mechanism and specific effects on bone metabolism have not been elucidated. In this study, we used rat bone marrow‐derived mesenchymal stem cells and found that imperatorin can activate RUNX2, COL1A1 and osteocalcin by promoting the Ser9 phosphorylation of GSK3β and entry of β‐catenin into the nucleus. Imperatorin also enhanced the production of phospho‐AKT (Ser473), an upstream factor that promotes the Ser9 phosphorylation of GSK3β. We used ipatasertib, a pan‐AKT inhibitor, to inhibit the osteogenic effect of imperatorin, and found that imperatorin promotes osteogenesis via AKT/GSK3β/β‐catenin pathway. Next, we used rat bone marrow‐derived monocytes, to check whether imperatorin inhibits osteoclast differentiation via AKT/GSK3β/β‐catenin pathway. Further, we removed the bilateral ovaries of rats to establish an osteoporotic model. Intragastric administration of imperatorin promoted osteogenesis and inhibited osteoclast in vivo. Our experiments showed that imperatorin is a potential drug for osteoporosis treatment.     

LPS administration increases CD11b<Superscript>+</Superscript> c-Fms<Superscript>+</Superscript> CD14<Superscript>+</Superscript> cell population that possesses osteoclast differentiation potential in mice

Osteoclasts are multinucleated giant cells that originate from a monocyte/macrophage lineage, and are involved in the inflammatory bone destruction accompanied by periodontitis. Recent studies have shown that osteoclast precursors reside not only in the bone marrow, but also in the peripheral blood and spleen, though the precise characteristics of each precursor have not been analyzed. We hypothesized that the number of osteoclast precursors in those tissues may increase under pathological conditions and contribute to osteoclast formation in vivo in a mouse model. To test this hypothesis, we attempted to identify cell populations that possess osteoclast differentiation potential in the bone marrow, spleen, and blood by analyzing macrophage/monocyte-related cell surface markers such as CD11b, CD14, and colony-stimulating factor-1 receptor (c-Fms). In the bone marrow, the CD11b^? cell population, but not the CD11b⁺ cell population, differentiated into osteoclasts in the presence of receptor activator of nuclear factor-κB ligand and macrophage colony-stimulating factor. On the other hand, in the spleen and blood, CD11b⁺ cells differentiated into osteoclasts. Interestingly, lipopolysaccharide (LPS) administration to the mice dramatically increased the proportion of CD11b⁺ c-Fms⁺ CD14⁺ cells, which differentiated into osteoclasts, in the bone marrow and spleen. These results suggest that LPS administration increases the proportion of a distinct cell population expressing CD11b⁺, c-Fms⁺, and CD14⁺ in the bone marrow and spleen. Thus, these cell populations are considered to contribute to the increase in osteoclast number during inflammatory bone destruction such as periodontitis.     

TET‐catalyzed oxidation of intragenic 5‐methylcytosine regulates CTCF‐dependent alternative splicing

Intragenic 5‐methylcytosine and CTCF mediate opposing effects on pre‐mRNA splicing: CTCF promotes inclusion of weak upstream exons through RNA polymerase II pausing, whereas 5‐methylcytosine evicts CTCF, leading to exon exclusion. However, the mechanisms governing dynamic DNA methylation at CTCF‐binding sites were unclear. Here, we reveal the methylcytosine dioxygenases TET1 and TET2 as active regulators of CTCF‐mediated alternative splicing through conversion of 5‐methylcytosine to its oxidation derivatives. 5‐hydroxymethylcytosine and 5‐carboxylcytosine are enriched at an intragenic CTCF‐binding sites in the CD45 model gene and are associated with alternative exon inclusion. Reduced TET levels culminate in increased 5‐methylcytosine, resulting in CTCF eviction and exon exclusion. In vitro analyses establish the oxidation derivatives are not sufficient to stimulate splicing, but efficiently promote CTCF association. We further show genomewide that reciprocal exchange of 5‐hydroxymethylcytosine and 5‐methylcytosine at downstream CTCF‐binding sites is a general feature of alternative splicing in naïve and activated CD4⁺ T cells. These findings significantly expand our current concept of the pre‐mRNA “splicing code” to include dynamic intragenic DNA methylation catalyzed by the TET proteins.     

Cell fusions in mammals

Cell fusions are important to fertilization, placentation, development of skeletal muscle and bone, calcium homeostasis and the immune defense system. Additionally, cell fusions participate in tissue repair and may be important to cancer development and progression. A large number of factors appear to regulate cell fusions, including receptors and ligands, membrane domain organizing proteins, proteases, signaling molecules and fusogenic proteins forming alpha-helical bundles that bring membranes close together. The syncytin family of proteins represent true fusogens and the founding member, syncytin-1, has been documented to be involved in fusions between placental trophoblasts, between cancer cells and between cancer cells and host cells. We review the literature with emphasis on the syncytin family and propose that syncytins may represent universal fusogens in primates and rodents, which work together with a number of other proteins to regulate the cell fusion machinery.     

Development of a LytE‐based high‐density surface display system in Bacillus subtilis

The three N‐terminal, tandemly arranged LysM motifs from a Bacillus subtilis cell wall hydrolase, LytE, formed a cell wall‐binding module. This module, designated CWBM_LytE, was demonstrated to have tight cell wall‐binding capability and could recognize two classes of cell wall binding sites with fivefold difference in affinity. The lower‐affinity sites were approximately three times more abundant. Fusion proteins with β‐lactamase attached to either the N‐ or C‐terminal end of CWBM_LytE showed lower cell wall‐binding affinity. The number of the wall‐bound fusion proteins was less than that of CWBM_LytE. These effects were less dramatic with CWBM_LytE at the N‐terminal end of the fusion. Both CWBM_LytE and β‐lactamase were essentially functional whether they were at the N‐ or C‐terminal end of the fusion. In the optimal case, 1.2 × 10⁷ molecules could be displayed per cell. As cells overproducing CWBM_LytE and its fusions formed filamentous cells (with an average of nine individual cells per filamentous cell), 1.1 × 10⁸β‐lactamase molecules could be displayed per filamentous cell. Overproduced CWBM_LytE and its fusions were distributed on the entire cell surface. Surface exposure and accessibility of these proteins were confirmed by immunofluorescence microscopy.     

Bone morphogenic protein 2 directly enhances differentiation of murine osteoclast precursors

Previous studies found that bone morphogenic proteins (BMPs) support osteoclast formation, but it is not clear whether this is a direct effect on osteoclasts or mediated indirectly through osteoblasts. We have shown that a mouse deficient for the BMP antagonist Twisted gastrulation suggested a direct positive role for BMPs on osteoclastogenesis. In this report, we further determine the significance of BMP signaling on osteoclast formation in vitro. We find that BMP2 synergizes with suboptimal levels of receptor activator of NF‐κB ligand (RANKL) to enhance in vitro differentiation of osteoclast‐like cells. The enhancement by BMP2 is not a result of changes in the rate of proliferation or survival of the bone marrow‐derived cultures, but is accompanied by an increase in expression of genes involved in osteoclast differentiation and fusion. Treatment with BMP2 did not significantly alter expression of RANKL or OPG in our osteoclast cultures, suggesting that the enhancement of osteoclastogenesis is not mediated indirectly through osteoblasts or stromal cells. Consistent with this, we detected phosphorylated SMAD1,5,8 (p‐SMAD) in the nuclei of mononuclear and multinucleated cells in osteoclast cultures. Levels of p‐SMAD, BMP2, and BMP receptors increased during differentiation. RNAi suppression of Type II BMP receptor inhibited RANKL‐stimulated formation of multinuclear TRAP‐positive cells. The BMP antagonist noggin inhibited RANKL‐mediated osteoclast differentiation when added prior to day 3, while addition of noggin on day 3 or later failed to inhibit their differentiation. Taken together, these data indicate that osteoclasts express BMP2 and BMP receptors, and that autocrine BMP signaling directly promotes the differentiation of osteoclasts‐like cells. J. Cell. Biochem. 109: 672–682, 2010. © 2009 Wiley‐Liss, Inc.     

LncRNA‐mRNA expression profiles and functional networks in osteoclast differentiation

Human osteoclasts are differentiated from CD14⁺ monocytes and are responsible for bone resorption. Long non‐coding RNAs (lncRNAs) have been proved to be significantly involved in multiple biologic processes, especially in cell differentiation. However, the effect of lncRNAs in osteoclast differentiation is less appreciated. In our study, RNA sequencing (RNA‐seq) was used to identify the expression profiles of lncRNAs and mRNAs in osteoclast differentiation. The results demonstrated that expressions of 1117 lncRNAs and 296 mRNAs were significantly altered after osteoclast differentiation. qRT‐PCR assays were performed to confirm the expression profiles, and the results were almost consistent with the RNA‐seq data. GO and KEGG analyses were used to predict the functions of these differentially expressed mRNA and lncRNAs. The Path‐net analysis demonstrated that MAPK pathway, PI3K‐AKT pathway and NF‐kappa B pathway played important roles in osteoclast differentiation. Co‐expression networks and competing endogenous RNA networks indicated that ENSG00000257764.2‐miR‐106a‐5p‐TIMP2 may play a central role in osteoclast differentiation. Our study provides a foundation to further understand the role and underlying mechanism of lncRNAs in osteoclast differentiation, in which many of them could be potential targets for bone metabolic disease.     

Purification of CD47‐streptavidin fusion protein from bacterial lysate using biotin–agarose affinity chromatography

CD47 is a widely expressed transmembrane glycoprotein that modulates the activity of a plethora of immune cells via its extracellular domain. Therefore, CD47 plays important roles in the regulation of immune responses and may serve as targets for the development of immunotherapeutic agents. To make sure CD47 functionality is intact under the process of protein conjugation, CD47‐streptavidin fusion protein was expressed and purified because it can easily bind to biotin‐tagged materials via the unique biotin–streptavidin affinity. In this study, gene sequences of CD47 extracellular domain (CD47ECD) and core streptavidin (coreSA) with a total 834 bp were inserted into pET20b plasmid to construct recombinant plasmid encoding CD47‐SA fusion gene. After bacteria transformation, the CD47‐SA fusion protein was expressed by isopropyl‐β‐d ‐thiogalactopyranoside (IPTG) induction. The collected bacteria lysate was loaded on biotinylated agarose to proceed the purification of CD47‐SA fusion protein. Due to the unexpected high affinity between biotin and coreSA, standard washing and elution approaches (e.g., varying pH, using biotin, and applying guanidine hydrochloride) reported for biotin–streptavidin affinity chromatography were not able to separate the target fusion protein. Instead, using low concentration of the non‐ionic detergent Triton X‐100 followed with alkaline buffer could efficiently weaken the binding between biotin and coreSA, thereby eluting out CD47‐SA fusion protein from the biotin agarose column. The purified CD47‐SA fusion protein was further characterized by molecular biology methods and its antiphagocytic functionality was confirmed by the phagocytosis assay. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:949–958, 2016