Recombinant expression, purification, and characterization of full-length human BST-2 from Escherichia coli.

HIV-1 virus release from infected cells is blocked by human BST-2, but HIV-1 Vpu efficiently antagonises BST-2 due to direct transmembrane domain interactions that occur between each protein. Targeting the interaction between these two proteins is seen as viable for HIV-1 antiviral intervention. This study describes the successful over-expression and purification of a recombinant full-length human BST-2 from inclusion bodies using affinity and anion exchange chromatography. Two milligrams of purified full-length BST-2 were produced per litre of BL21 (DE3) T7 Express® pLysY E. coli culture. Far-UV circular dichroism validated the renaturing of the recombinant protein and retention of its secondary structure. Furthermore, through ELISA, a known human BST-2 binding partner, HIV-1 Vpu, was shown to bind to the renatured and purified protein, further validating its folding. To our knowledge this is the first report of the purification of a wild-type, full-length human BST-2 from Escherichia coli.

Determination of CD43 and CD200 surface expression improves accuracy of B-cell lymphoma immunophenotyping.

BACKGROUND: The Matutes score (MS) was proposed to differentiate chronic lymphocytic leukemia (CLL) from other B-cell non-Hodgkin lymphomas (B-NHLs). However, ambiguous immunophenotypes are common and remain a diagnostic challenge. Therefore, we evaluated the diagnostic benefit of measuring CD200 and CD43 expression together with the standard MS antigens. METHODS: 138 lymphoma patient samples and a validation cohort of 138 additive samples were classified according to the standard MS and further assigned with one or two additional points, for high CD200 and/or CD43 expression levels. The "classical" MS and the "Matutes score-extended" (MS-e) were categorized as high (4-5/6-7), intermediate (2-3/4-5), and low (0-1/0-3). Samples were reclassified into the MS-e with focus on ambiguous cases with an intermediate "classical" MS. RESULTS: A total of 35 of 138 (25.4%) patient samples were assigned to the intermediate MS group and confirmed by histopathological reports as CLL (14/40.0%) and B-NHLs other than CLL (21/60%). MS-e analysis identified 13 of 14 (92.9%) of CLL cases (MS-e 4-5) and 18/21 (85.7%) non-CLL cases (MS-e ≤ 3) correctly. Overall, the sensitivity of the CLL diagnosis was significantly increased by application of MS-e compared to the "classical" MS (98.8% vs. 82.7%; p = 0.0009), while specificity of both methods was almost equal (94.7% vs. 98.3%; p = 0.4795). Of note, sole measurement of CD43 and CD200 on B-cells sufficiently differentiated CLL from non-CLL with a test accuracy superior to the "classical" MS (F1 score 96.2 vs. 93.6). CONCLUSION: CD200 and CD43 have a high informative value in diagnostic immunophenotyping and facilitate the separation of CLL from other B-NHLs particularly in ambiguous cases.

The Orphan Immune Receptor LILRB3 Modulates Fc Receptor-Mediated Functions of Neutrophils.

Neutrophils are critical to the generation of effective immune responses and for killing invading microbes. Paired immune receptors provide important mechanisms to modulate neutrophil activation thresholds and effector functions. Expression of the leukocyte Ig-like receptor (LILR)A6 (ILT8/CD85b) and LILRB3 (ILT5/CD85a) paired-receptor system on human neutrophils has remained unclear because of the lack of specific molecular tools. Additionally, there is little known of their possible functions in neutrophil biology. The objective of this study was to characterize expression of LILRA6/LILRB3 receptors during human neutrophil differentiation and activation, and to assess their roles in modulating Fc receptor-mediated effector functions. LILRB3, but not LILRA6, was detected in human neutrophil lysates following immunoprecipitation by mass spectrometry. We demonstrate high LILRB3 expression on the surface of resting neutrophils and release from the surface following neutrophil activation. Surface expression was recapitulated in a human PLB-985 cell model of neutrophil-like differentiation. Continuous ligation of LILRB3 inhibited key IgA-mediated effector functions, including production of reactive oxygen species, phagocytic uptake, and microbial killing. This suggests that LILRB3 provides an important checkpoint to control human neutrophil activation and their antimicrobial effector functions during resting and early-activation stages of the neutrophil life cycle.

An electrochemical biosensor for the detection of epithelial-mesenchymal transition.

Epithelial-mesenchymal transition (EMT) is critically involved in a variety of biological processes. Electrochemical sensing offers potential to develop more effective technology for EMT detection. In this study, by using the unique performance of quantum dot (QD)-nanocomposite materials, we establish an electrochemical biosensor that can specifically detect the change of E-cadherin and analyze different stages of EMT. The signal for EMT is largely magnified due to the transmission of molecular information to the electronic device. In addition, differential pulse voltammetry reveals that the response of the electrochemical signals is rapid and sensitive, due to the synergistic effect of QDs and carbon nanotube-gold nanoparticles. Our study thus suggests that electrochemical sensing is an effective technology for detecting EMT and may have broad applications in analyzing various cell type transitions.

Langerhans cell histiocytosis of a metatarsal bone in an adult female.

Langerhans cell histiocytosis (LCH) commonly occurs in children. It mimics infection and many benign and malignant tumours. This disease mainly involves the spine, skull and long bones, and its incidence is sporadic in the small bones of the foot and hand. We could not find any case reports with the involvement of a metatarsal bone, and hence, awareness about its possibility is essential to suspect it as a differential diagnosis of lytic lesions in the foot bones and therefore treat it judiciously. We have reported a case of a 35-year-old woman with spontaneous onset of pain over her right foot for the last year. An extensive curettage was performed, where the histology confirmed the features of LCH. Awareness about this entity and its differential diagnosis may help to clinch and early diagnosis and to treat effectively.

Schiff bases of tryptamine as potent inhibitors of nucleoside triphosphate diphosphohydrolases (NTPDases): Structure-activity relationship.

Overexpression of NTPDases leads to a number of pathological situations such as thrombosis, and cancer. Thus, effective inhibitors are required to combat these pathological situations. Different classes of NTPDase inhibitors are reported so far including nucleotides and their derivatives, sulfonated dyes such as reactive blue 2, suramin and its derivatives, and polyoxomatalates (POMs). Suramin is a well-known and potent NTPDase inhibitor, nonetheless, a range of side effects are also associated with it. Reactive blue 2 also had non-specific side effects that become apparent at high concentrations. In addition, most of the NTPDase inhibitors are high molecular weight compounds, always required tedious chemical steps to synthesize. Hence, there is still need to explore novel, low molecular weight, easy to synthesize, and potent NTPDase inhibitors. Keeping in mind the known NTPDase inhibitors with imine functionality and nitrogen heterocycles, Schiff bases of tryptamine, 1-26, were synthesized and characterized by spectroscopic techniques such as EI-MS, HREI-MS, 1H-, and 13C NMR. All the synthetic compounds were evaluated for the inhibitory avidity against activities of three major isoforms of NTPDases: NTPDase-1, NTPDase-3, and NTPDase-8. Cumulatively, eighteen compounds were found to show potent inhibition (Ki = 0.0200-0.350 µM) of NTPDase-1, twelve (Ki = 0.071-1.060 µM) of NTPDase-3, and fifteen compounds inhibited (Ki = 0.0700-4.03 µM) NTPDase-8 activity. As a comparison, the Kis of the standard inhibitor suramin were 1.260 ±â€¯0.007, 6.39 ±â€¯0.89 and 1.180 ±â€¯0.002 µM, respectively. Kinetic studies were performed on lead compounds (6, 5, and 21) with human (h-) NTPDase-1, -3, and -8, and Lineweaver-Burk plot analysis showed that they were all competitive inhibitors. In silico study was conducted on compound 6 that showed the highest level of inhibition of NTPDase-1 to understand the binding mode in the active site of the enzyme.

Development of a selective and highly sensitive fluorescence assay for nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39).

Ecto-nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) is a major ectonucleotidase that hydrolyzes proinflammatory ATP via ADP to AMP, which is subsequently converted by ecto-5'-nucleotidase (CD73) to immunosuppressive adenosine. Activation of CD39 has potential for treating inflammatory diseases, while inhibition was suggested as a novel strategy for the immunotherapy of cancer. In the present study, we developed a selective and highly sensitive capillary electrophoresis (CE) assay using a novel fluorescent CD39 substrate, a fluorescein-labelled ATP (PSB-170621A) that is converted to its AMP derivative. To accelerate the assays, a two-directional (forward and reverse) CE system was implemented using 96-well plates, which is suitable for the screening of compound libraries (Z'-factor: 0.7). The detection limits for the forward and reverse operation were 11.7 and 2.00 pM, respectively, indicating a large enhancement in sensitivity as compared to previous methods (e.g. malachite-green assay: 1 000 000-fold, CE-UV assay: 500 000-fold, fluorescence polarization immunoassay: 12 500-fold). Enzyme kinetic studies at human CD39 revealed a Km value of 19.6 µM, and a kcat value of 119 × 10-3 s-1 for PSB-170621A, which shows similar substrate properties as ATP (11.4 µM and 82.5 × 10-3 s-1). The compound displayed similar properties at rat and mouse CD39. Subsequent docking studies into a homology model of human CD39 revealed a hydrophobic pocket that accommodates the fluorescein tag. PSB-170621A was found to be preferably hydrolyzed by CD39 as compared to other ectonucleotidases. The new assay was validated by performing inhibition assays with several standard CD39 inhibitors yielding results that were consonant with data using the natural substrates.

Sialylated keratan sulfate proteoglycans are Siglec-8 ligands in human airways.

Human siglecs are a family of 14 sialic acid-binding proteins, most of which are expressed on subsets of immune cells where they regulate immune responses. Siglec-8 is expressed selectively on human allergic inflammatory cells-primarily eosinophils and mast cells-where engagement causes eosinophil apoptosis and inhibits mast cell mediator release. Evidence supports a model in which human eosinophils and mast cells bind to Siglec-8 sialoglycan ligands on inflammatory target tissues to resolve allergic inflammation and limit tissue damage. To identify Siglec-8-binding sialoglycans from human airways, proteins extracted from postmortem human trachea were resolved by size-exclusion chromatography and composite agarose-acrylamide gel electrophoresis, blotted and probed by Siglec-8-Fc blot overlay. Three size classes of Siglec-8 ligands were identified: 250 kDa, 600 kDa and 1 MDa, each of which was purified by affinity chromatography using a recombinant pentameric form of Siglec-8. Proteomic mass spectrometry identified all size classes as the proteoglycan aggrecan, a finding validated by immunoblotting. Glycan array studies demonstrated Siglec-8 binding to synthetic glycans with a terminal Neu5Acα2-3(6-sulfo)-Gal determinant, a quantitatively minor terminus on keratan sulfate (KS) chains of aggrecan. Treating human tracheal extracts with sialidase or keratanase eliminated Siglec-8 binding, indicating sialylated KS chains as Siglec-8-binding determinants. Treating human tracheal histological sections with keratanase also completely eliminated the binding of Siglec-8-Fc. Finally, Siglec-8 ligand purified from human trachea extracts induced increased apoptosis of freshly isolated human eosinophils in vitro. We conclude that sialylated KS proteoglycans are endogenous human airway ligands that bind Siglec-8 and may regulate allergic inflammation.

Transcriptional Output Transiently Spikes Upon Mitotic Exit.

The pulsatile nature of gene activity has recently emerged as a general property of the transcriptional process. It has been shown that the frequency and amplitude of transcriptional bursts can be subjected to extrinsic regulation. Here we have investigated if these parameters were constant throughout the cell cycle using the single molecule RNA FISH technique. We found evidence of transcriptional spikes upon mitotic exit in three different human cell lines. Recording of cell growth prior to hybridization and immuno-RNA FISH analysis revealed that these spikes were short-lived and subsided before completion of cytokinesis. The transient post-mitotic increase in transcriptional output was found to be the result of cells displaying a higher number of active alleles and/or an increased number of nascent transcripts per active allele, indicating that both the burst fraction and the amplitude of individual bursts can be increased upon mitotic exit. Our results further suggest that distinct regulatory mechanisms are at work shortly after mitotic exit and during the rest of interphase. We speculate that transcriptional spikes are associated with chromatin decondensation, a hallmark of post-mitotic cells that might alter the dynamics of transcriptional regulators and effectors.

Expression and purification of functional insulin and insulin-like growth factor 1 holoreceptors from mammalian cells.

The insulin receptor (IR) and insulin-like growth factor 1 receptor (IGF1R) are receptor tyrosine kinases (RTKs) involved in the regulation of many important cellular processes. The current proposed models of activation are derived from structural studies using soluble extracellular domains and cytoplasmic tyrosine kinase domains. Preparations of full length IR and IGF1R have been hampered by the need for unconventional affinity chromatography resins and/or harsh eluting conditions. Here, we present a purification protocol to obtain full-length, detergent solubilized IR and IGF1R at quantities suitable for biochemical and structural characterization. We screened a panel of 24 structurally diverse detergents for optimal ligand activation. The receptors purified in n-dodecyl-ß-D-maltoside showed ligand-stimulated autophosphorylation and kinase activity, suggesting an intact transmembrane signaling mechanism. This convenient purification protocol can be used to produce high quantities of IR, IGF1R, or other RTKs, and can be adapted for other challenging membrane proteins.

Basophil activation test: Implementation and standardization between systems and between instruments.

The basophil activation test (BAT) is a good ex vivo alternative for measuring hypersensitivity to an allergen in sensitized patients but still lacks standardization. In this present study, we have implemented one of the systems and proposed inter-systems, inter-instrument standardization. Our method for basophil activation and labeling on whole blood: EDTA in one step using BasoflowEx® and FlowCast® . Setup on Navios and fluorescence targets converted to set up FACSCanto™ instrument. Our results: 1) A CD203c/CD63 (BasoflowEx) method was adapted for EDTA samples and simplified. 2) Final washing and concentration and use of time parameter help acquiring as many basophils as possible, spare acquisition time and noise. 3) The modified method was validated according to ISO15189 with a precision at 5.1% RCV, linearity between 1 and 1/8 of anti-IgE stimulation. Results were very close with CCR3/CD63 system (FlowCast). 4) Standardization, between systems and even between instruments. Mean Fluorescence Intensity targets are proposed using standard beads (Cytocal® ) middle peak: FITC = 19.4; PE = 28.8 on Navios® corresponding to FITC = 4,966; PE = 7,373 for FACSCanto. Data analyzed on common software (Kaluza® ) were very closely correlated. 5) Co-labeling of B cells (CD20+) gives the possibility to monitor a significant drop of basophils under stimulation that could explain some underestimation in case of strong hypersensitivity. In conclusion, BAT would strongly benefit from easy implementation [EDTA, one step stimulation/labeling, wash, full sample analysis over time parameter, B cell relative basophil count] and standardization of instrument settings on MFI targets whatever system or instrument is used. © 2017 International Society for Advancement of Cytometry.

The expression of NTPDase1 and -2 of Leishmania infantum chagasi in bacterial and mammalian cells: Comparative expression, refolding and nucleotidase characterization.

Visceral Leishmaniasis (VL) represents an important global health problem in several warm countries around the world. The main targets in this study are the two nucleoside triphosphate diphosphohydrolases (NTPDases) from Leishmania infantum chagasi that are the main etiologic agent of VL in the New World. These enzymes, called LicNTPDase1 and -2, are homologous to members 5 and 6 of the mammalian E-NTPDase/CD39 superfamily of enzymes. These enzymes hydrolyze nucleotides and accordingly can participate in the purine salvage pathways and in the modulation of purinergic signaling through the extracellular nucleotide-dependent host immune responses. They can therefore affect adhesion and infection of host cells and the parasite virulence. To further characterize these enzymes, in this work, we expressed LicNTPDase1 and -2 in the classical bacterial system Escherichia coli and mammalian cell system COS-7 cells. Our data demonstrate that changes in refolding after expression in bacteria can increase the activity of recombinant (r) rLicNTPDase2 up to 20 times but has no significant effect on rLicNTPDase1. Meanwhile, the expression in COS-7 led to a significant increase in activity for rLicNTPDase1.

Isolating Fc-Tagged SEMA4D Recombinant Protein from 293FT Cells.

Recombinant proteins are widely used in biomedical sciences, and in pharmaceutical research. Through genetic recombination, specific DNA sequences are engineered and inserted into a biological host. Once inside the host, the DNA is transcribed and translated into the target protein and is ready to be purified. Here, we describe the transfection, purification, and visualization of Fc-tagged SEMA4D (semaphorin 4D) recombinant protein.

Identification of CD200+ colorectal cancer stem cells and their gene expression profile.

CD200 is a cell surface glycoprotein that has been implicated in a variety of human cancer cells. It has been proposed as a cancer stem cell (CSC) marker in colon cancer and is closely related to tumor immunosuppression. However, there is little functional data supporting its role as a true CSC marker, and the mechanism by which CD200 contributes to colorectal cancer has not been elucidated. In the present study, CD200+ and CD200- COLO 205 colorectal cancer cells were sorted out by flow cytometry, and colonosphere formation and Transwell migration assays were performed. Affymetrix Human U133 Plus2.0 arrays were used to screen the gene expression profiles of CD200+ and CD200- colorectal cancer cells. The results suggest that there are differentially expressed genes between the two subpopulations, including several important genes that function in cell proliferation, metastasis, apoptosis and the immune response. Pathway analysis revealed that the Wnt, MAPK and calcium signaling pathways were differentially expressed between CD200+ and CD200- cells. Moreover, several key genes upregulated in CD200+ cells were also highly overexpressed in CD44+CD133+ colorectal stem cells compared to the CD44-CD133- fraction of the same cell line. In the present study, we showed for the first time a correlation between CD200 expression and the Wnt signaling pathway in colon cancer cells.

Generation of Rat Monoclonal Antibodies Against Human Pancreatic Ductal Adenocarcinoma Cells.

Pancreatic ductal adenocarcinoma is an aggressive tumor with a poor prognosis. Biomarkers that can detect the tumor in its early stages when it may be amenable to curative resection might improve prognosis. To discover novel markers expressed in primary pancreatic cancer, we generated a panel of monoclonal antibodies against pancreatic ductal adenocarcinoma cell line BxPC3 using a rat medial iliac lymph node method. The antigen recognized by 1B5A5 was expressed on the cell surface and secreted into the conditioned medium of BxPC3 cells, and characterized as glycoproteins with molecular mass between 60 and 90 kDa. A wide range of molecular weights of 1B5A5 antigen in several pancreatic cancer cell lines were observed. Immunohistochemistry using a human multiple organ tumor tissue array showed an enhanced expression of 1B5A5 antigen in pancreas, lung, stomach, breast, urinary bladder, colon, and cervix uteri cancers. Immunoprecipitation followed by proteomic analyses identified CEACAM6 as a 1B5A5 antigen. In addition, western blot analysis results indicated that the 1B5A5 epitope is located within an amino-terminal domain of CEACAM6. These results raised the possibility that our approach could lead to discovery of novel biomarkers for the early stage of cancers in a relatively short period of time.

Lymphocyte depletion and repopulation after chemotherapy for primary breast cancer.

BACKGROUND: Approximately 30 % of breast cancer patients receive chemotherapy, yet little is known about influences of current regimens on circulating lymphocyte levels and phenotypes. Similarly, clinico-pathological factors that modify these influences, and implications for future immune health remain mainly unexplored. METHODS: We used flow-cytometry to assess circulating lymphocyte levels and phenotypes in 88 primary breast cancer patients before chemotherapy and at time-points from 2 weeks to 9 months after chemotherapy completion. We examined circulating titres of antibodies against pneumococcal and tetanus antigens using ELISAs. RESULTS: Levels of B, T and NK cells were significantly reduced 2 weeks after chemotherapy (p < 0.001). B cells demonstrated particularly dramatic depletion, falling to 5.4 % of pre-chemotherapy levels. Levels of all cells recovered to some extent, although B and CD4(+) T cells remained significantly depleted even 9 months post-chemotherapy (p < 0.001). Phenotypes of repopulating B and CD4(+) T cells were significantly different from, and showed no sign of returning to pre-chemotherapy profiles. Repopulating B cells were highly depleted in memory cells, with proportions of memory cells falling from 38 % to 10 % (p < 0.001). Conversely, repopulating CD4(+) T cells were enriched in memory cells, which increased from 63 % to 75 % (p < 0.001). Differences in chemotherapy regimen and patient smoking were associated with significant differences in depletion extent or repopulation dynamics. Titres of anti-pneumococcal and anti-tetanus antibodies were both significantly reduced post-chemotherapy and did not recover during the study (p < 0.001). CONCLUSION: Breast cancer chemotherapy is associated with long-term changes in immune parameters that should be considered during clinical management.

Saturation monitoring of VX15/2503, a novel semaphorin 4D-specific antibody, in clinical trials.

BACKGROUND: Receptor occupancy, or saturation, assays are often utilized in preclinical and clinical development programs to evaluate the binding of a biologic to a cellular target. These assays provide critical information regarding the dose of drug required to "saturate" the target as well as important pharmacodymamic (PD) data. A flow cytometric method was developed to measure the degree of Semaphorin 4D (SEMA4D; CD100) saturation by VX15/2303, an investigational monoclonal antibody specific for SEMA4D. METHODS: The assay detects VX15/2503, a human IgG4 specific for SEMA4D, with an IgG4 -specific monoclonal antibody. RESULTS: Data generated allowed assessment of two related SEMA4D-specific pharmacodynamic (PD) markers: (1) The measurement of cellular SEMA4D (cSEMA4D) saturation by VX15/2503, and (2) the cell membrane expression levels of cSEMA4D. CONCLUSIONS: This assay specifically and reproducibly measured cSEMA4D saturation and expression levels. Evaluation of the SEMA4D-specific PD markers were critical in determining the clinical saturation threshold of cSEMA4D by VX15/2503.

Molecular Pathology of Adult T-Cell Leukemia/Lymphoma.

Adult T-cell leukemia/lymphoma (ATLL) is a peripheral T-cell neoplasm of highly pleomorphic lymphoid cells. ATLL is usually widely disseminated, and it is caused by human T-cell leukemia virus type 1 (HTLV-1). It is a disease with a long latency, and affected individuals are usually exposed to the virus very early in life. The cumulative incidence of ATLL is estimated to be 2.5% among HTLV-1 carriers. ATLL cells express CD2, CD3, CD5, CD4, and CD25, as well as CCR4 and FoxP3 of the regulatory T-cell marker. HTLV-1 is causally linked to ATLL, but infection alone is not sufficient to result in neoplastic transformation. A significant finding in this connection is that the Tax viral protein leads to transcriptional activation of many genes, while the HTLV-1 basic leucine zipper factor is thought to be important for T-cell proliferation and oncogenesis. Half of ATLL cases retain the ability to express HTLV-1 Tax, which is a target of HTLV-1-specific cytotoxic T lymphocytes (CTL). An increase in HTLV-1-specific CTL responses is observed in some asymptomatic HTLV-1 carriers. Although HTLV-1-specific CTL are also present in the peripheral blood of ATLL patients, they do not expand sufficiently. We investigated the clinicopathological features and analyzed the staining of Tax-specific CTL and FoxP3. Tax-specific CTL correlated inversely with FoxP3, an increase in the ratio of CD163+ tumor-associated macrophages was associated with worse clinical prognosis, and ATLL cell lines proliferated significantly following direct co-culture with M2 macrophages. Several clinical variants of ATLL have been identified: acute, lymphomatous, chronic, and smoldering. Oligo-array comparative genomic hybridization revealed that genomic loss of 9p21.3 was a significant characteristic of acute-type, but not of chronic-type ATLL. Furthermore, we found that genomic alteration of CD58, which is implicated in immune escape, is more frequently observed in acute than in chronic ATLL. Interestingly, the chronic cases with cell cycle deregulation and disruption of immunosurveillance mechanism were associated with faster progression to acute ATLL. Immune evasion, microenvironment, and genetic alteration are therefore important in the multi-step progression of ATLL lymphomagenesis.

Proteomics characterization of exosome cargo.

Characterization of exosomal cargo is of significant interest because this cargo can provide clues to exosome biogenesis, targeting, and cellular effects and may be a source of biomarkers for disease diagnosis, prognosis and response to treatment. With recent improvements in proteomics technologies, both qualitative and quantitative characterization of exosomal proteins is possible. Here we provide a brief review of exosome proteomics studies and provide detailed protocols for global qualitative, global quantitative, and targeted quantitative analysis of exosomal proteins. In addition, we provide an example application of a standard global quantitative analysis followed by validation via a targeted quantitative analysis of urine exosome samples from human patients. Advantages and limitations of each method are discussed as well as future directions for exosome proteomics analysis.

A convenient method for hTfR1 inclusion body purification.

Human transferrin receptor, referred as hTfR1, is ubiquitously expressed at low levels in most normal human tissues; however, the expression level of hTfR1 at the blood-brain barrier (BBB) and in tumor tissues is relatively higher. hTfR1 is a type II homodimeric transmembrane protein. The extracellular domain of hTfR1 consists of three domains: helical domain, apical, and protease-like domain. In order to prepare hTfR1 antibody, which can be utilized to deliver drugs across BBB through receptor-mediated endocytosis, we began to express the nonligand binding domain of hTfR1 in Escherichia coli BL21 Transetta (DE3). The TfR1 gene was first obtained from HepG2 cells by reverse-transcription polymerase chain reaction (RT-PCR) and then inserted into pET 32a(c+) vector. The protein was expressed in the form of inclusion body with extremely high purity by the E. coli BL21 Transetta (DE3), and the purity was further improved by size-exclusion chromatography. The Western blot test indicated that the recombinant protein was TfR1 as expected. Above all, this report provided a convenient protocol that could be fulfilled in order to prepare hTfR1 inclusion body, which failed to be purified by an Ni(2+) affinity column.