Multimodal CEA-Targeted Image-Guided Colorectal Cancer Surgery using 111In-Labeled SGM-101.

PURPOSE: Intraoperative image guidance may aid in clinical decision-making during surgical treatment of colorectal cancer. We developed the dual-labeled carcinoembryonic antigen-targeting tracer, [111In]In-DTPA-SGM-101, for pre- and intraoperative imaging of colorectal cancer. Subsequently, we investigated the tracer in preclinical biodistribution and multimodal image-guided surgery studies, and assessed the clinical feasibility on patient-derived colorectal cancer samples, paving the way for rapid clinical translation. EXPERIMENTAL DESIGN: SGM-101 was conjugated with p-isothiocyanatobenzyl-diethylenetriaminepentaacetic acid (DTPA) and labeled with Indium-111 (111In). The biodistribution of 3, 10, 30, and 100 µg [111In]In-DTPA-SGM-101 was assessed in a dose escalation study in BALB/c nude mice with subcutaneous LS174T human colonic tumors, followed by a study to determine the optimal timepoint for imaging. Mice with intraperitoneal LS174T tumors underwent micro-SPECT/CT imaging and fluorescence image-guided resection. In a final translational experiment, we incubated freshly resected human tumor specimens with the tracer and assessed the tumor-to-adjacent tissue ratio of both signals. RESULTS: The optimal protein dose of [111In]In-DTPA-SGM-101 was 30 µg (tumor-to-blood ratio, 5.8 ± 1.1) and the optimal timepoint for imaging was 72 hours after injection (tumor-to-blood ratio, 5.1 ± 1.0). In mice with intraperitoneal tumors, [111In]In-DTPA-SGM-101 enabled preoperative SPECT/CT imaging and fluorescence image-guided resection. After incubation of human tumor samples, overall fluorescence and radiosignal intensities were higher in tumor areas compared with adjacent nontumor tissue (P < 0.001). CONCLUSIONS: [111In]In-DTPA-SGM-101 showed specific accumulation in colorectal tumors, and enabled micro-SPECT/CT imaging and fluorescence image-guided tumor resection. Thus, [111In]In-DTPA-SGM-101 could be a valuable tool for preoperative SPECT/CT imaging and intraoperative radio-guided localization and fluorescence image-guided resection of colorectal cancer.

Analysis of the inhibiting activity of reversion-inducing cysteine-rich protein with Kazal motifs (RECK) on matrix metalloproteinases.

Matrix metalloproteinases (MMPs) occur in 23 human paralogues with key functions in physiology, and their activity is controlled by protein inhibitors. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), which is essential for embryogenesis and tumour suppression, has been reported to inhibit MMPs. Here, we developed eukaryotic and bacterial expression systems for different RECK variants and analysed their inhibitory capacity against representative MMPs in vitro. We could not detect any significant inhibition. Instead, we found that partially purified RECK from the conditioned medium of transfected Expi293F cells but not that of ExpiCHO-S or Drosophila Schneider cells contained a contaminant with proteolytic activity. The contaminant was removed through treatment with a small-molecule serine peptidase inhibitor and additional chromatographic purification. A tantamount contaminant was further detected in an equivalent expression system of the N-terminal fragment of the proteoglycan testican 3, but not in those of two other proteins. These results indicate that previous reports of inhibitory activity of recombinant RECK on MMPs, which were performed with partially purified samples, were probably masked by a coeluting contaminant present in the supernatant of HEK293-derived cells. Thus, RECK is probably not a direct inhibitor of MMP catalytic activity but may still regulate MMPs through other mechanisms.

Wrinkled metal based quantum sensor for In vitro cancer diagnosis.

This article presents a unique 3D biocompatible Aluminum-based quantum structure (QS) for in vitro cancer detection using Surface Enhanced Raman Scattering (SERS). The Al-based QSs fabricated using ultrashort pulsed laser are of two distinct surface characters, wrinkled and smooth spherical. The limit of detection for chemical sensing of Crystal Violet and Rhodamine 6G by the Al-QS was driven up to single molecule sensing (femtomolar concentration). Biological sensing of cysteine, a disease biomarker and carcinoembryonic antigen (CEA), a cancer biomarker was also tested by the Al-QS. The ability of in vitro cell detection using Al-QS was analyzed with three cell lines, mammalian fibroblast and pancreatic and lung cancer cells. The Al-QS were up taken by the cells through label-free self-internalization and were sensed by SERS. Further assay was performed to differentiate cancerous and non-cancerous cells by measuring lipid and protein peak intensity within the cells. The result of this research indicated that SERS based Al-QS could be a suitable candidate for the early diagnosis of cancer.

An Optical Microfiber Biosensor for CEACAM5 Detection in Serum: Sensitization by a Nanosphere Interface.

The detection of carcinoembryonic antigen (CEA)-related cell adhesion molecules 5 (CEACAM5) is significant in cancer prewarning. Early diagnosis can effectively alleviate the danger of cancer. Point-of-care testing (POCT) has become a competitive technology for early detection. Fiber optic biosensors have great potential as POCT tools. However, their limits of detection (LODs) are not sufficient to afford ultralow concentration detection at the early stage. Herein, this work presents an optical microfiber sensor functionalized by a polystyrene@gold nanosphere (PS@Au nanosphere) interface for a synergistic sensitization effect to detect the ultralow CEACAM5 concentrations in serum at the early stage. The sensor's LOD achieves 3.54 × 10-17 M in pure solution and 5.27 × 10-16 M in serum, with the sensitization effect coupled with surface area enlargement and electromagnetic enhancement of interface. This LOD is about 6 orders of magnitude lower than that of current methods. It can be employed to detect the biomarkers at ultralow concentrations present in serum in the early stages of cancer. As the interfacial synergistic sensitization strategy is suitable for refractive index (RI)-based optical transducers, this work provides new opportunities to employ fiber optic biosensors as effective POCT tools.

Mulberry-like Au@PtPd porous nanorods composites as signal amplifiers for sensitive detection of CEA.

Effective detection of cancer biomarkers plays a crucial role in the prevention of early cancer. Here, a sandwich-type electrochemical immunosensor was successfully constructed for sensitive detection of carcinoembryonic antigen (CEA) using MoS2/CuS-Au as sensing platform and mulberry-like Au@PtPd porous nanorods (Au@PtPd MPs) as signal amplifiers. The large surface area and good biocompatibility of MoS2/CuS-Au increased the loading of primary antibody. And the good conductivity of MoS2/CuS-Au accelerated the electron transport rate of the electrode surface. Au@PtPd MPs with large specific surface area and a large number of catalytically active sites showed excellent electrocatalytic performance for hydrogen peroxide reduction. The sandwich-type immunosensor prepared by the signal amplification strategy exhibited a wide linear detection range (50 fg/mL to 100 ng/mL) and a low detection limit of 16.7 fg/mL (S/N = 3), featuring good selectivity, stability and reproducibility. Moreover, the satisfactory results in analysis of human serum samples indicated that it possessed a potential application promising in early clinical diagnoses.

Resonance Raman scattering-infrared absorption dual-mode immunosensing for carcinoembryonic antigen based on ZnO@SiO2 nanocomposites.

Detection of cancer biomarkers is crucial for the diagnosis and monitoring of malignant tumors. However, the accuracy and sensitivity still require sufficient improvement for practically clinical application. In this work, a reliable and sensitive dual-mode immunosensing method is described for carcinoembryonic antigen (CEA) detection using a biofunctional ZnO@SiO2 nanocomposite as a resonance Raman scattering (RRS)-infrared (IR) absorption nanoprobe. The multiphonon RRS signal originating from the ZnO and the characteristic IR fingerprint signal of the transverse optical and longitudinal optical phonon modes of the asymmetric stretching of Si-O-Si bonds showed no interference with each other. A CEA antibodies-immobilized substrate was fabricated to capture the analyte/nanoprobe complexes. The RRS intensity at 569 cm‒1 and the IR absorption at 1061 cm‒1 were used for quantitative analysis. Accurate CEA detection was performed as a result of the strong resistance of the dual-mode nanoprobe to surrounding interference. The limit of detection was 98.0 fg mL‒1. The detection range was 500 ng mL‒1 - 50 fg mL‒1, which is wider than those of single-mode RRS or IR absorption immunosensings. High reproducibility, selectivity and specificity were achieved. The assay performance of human serum samples demonstrated the practicability of the method in clinical cancer diagnosis.

Orthogonal dual molecularly imprinted polymer-based plasmonic immunosandwich assay: A double characteristic recognition strategy for specific detection of glycoproteins.

Sensitive and specific detection methods are critical to the detection of glycoproteins. Immunoassay has been a powerful tool for this purpose, in which antibodies or their mimics particularly molecularly imprinted polymers (MIPs) are used for specific recognition. Epitope and glycan are two structure features of a glycoprotein. However, immunoassays based on simultaneous recognition towards the two characteristics have been scarcely explored so far. Herein we present a new strategy called orthogonal dual molecularly imprinted polymer-based plasmonic immunosandwich assay (odMIP-PISA). It relies on double recognition towards a target glycoprotein by two different types of MIPs, using epitope-imprinted gold nanoparticles (AuNPs)-coated slide as capturing substrate to recognize the peptide epitope and glycans-imprinted Raman-active silver nanoparticles as labeling nanotags to recognize the glycans. Carcinoembryonic antigen (CEA), a routinely used marker for colon cancer, was used as a test glycoprotein. The orthogonal double recognition apparently improved the specificity, reducing the maximum cross-reactivity from 14.4% for epitope recognition and 15.2% for glycan recognition to 8.2% for double recognition. Meanwhile, the plasmonic nanostructure-based Raman detection provided ultrahigh sensitivity, yielding a limit of detection of 5.56 × 10-14 M (S/N = 10). Through measuring the CEA level in human serum, this method permitted differentiation of colon cancer patient from healthy individual. Compared with the traditional immunoassay, odMIP-PISA exhibited multiple advantages, including simplified procedure (6 steps), speed (30 min), reduced cost, and so on. Therefore, this new approach holds great promise in many applications particularly clinical diagnosis.

Novel single-chain variant of antibody against mesothelin established by phage library.

Mesothelin (MSLN) shows increased expression in various cancer cells. For clinical application of antibodies as a positron emission tomography (PET) imaging reagent, a human shortened antibody is essential both for avoiding redundant immune responses and for providing rapid imaging. Therefore, we cloned a single-chain fragment of variable regions (scFv) from a human-derived gene sequence. This was achieved through the construction of a naïve phage library derived from human tonsil lymphocytes. Using a column with human recombinant MSLN, we carried out bio-panning of phage-variants by colony formation. We first obtained 120 clones that were subjected to selection in an ELISA using human recombinant MSLN as a solid phase antigen, and 15 phage clones of scFv with a different sequence were selected and investigated by flow cytometry (FCM). Then, six variants were selected and the individual scFv gene was synthesized in the VL and VH domains and expressed in Chinese hamster ovary cells. Mammalian cell-derived human-origin scFv clones were analyzed by FCM again, and one MSLN highly specific scFv clone was established. PET imaging by 89 Zr-labeled scFv was done in mice bearing xenografts with MSLN-expressing cancer cells, and tumor legions were successfully visualized. The scFv variant established in the present study may be potentially useful for cancer diagnosis by PET imaging.

Production of polyclonal antibody against human Neuritin and its application of immunodetection.

OBJECTIVE: To date, a commercial antibody to human Neuritin for immunoprecipitation is still limited. In this study, we aimed to develop a specific antibody for further research on the potential function of Neuritin. METHODS AND RESULTS: By epitope prediction of recombinant human Neuritin, the active fragment of human Neuritin that could be used as an excellent immunogen. Soluble His-tagged Neuritin was expressed and purified from Pichia pastoris. Polyclonal antibody against Neuritin was obtained by immunizing Sprague-Dawley rats with purified recombinant human Neuritin. Affinity-purified polyclonal antibody against Neuritin was characterized with indirect enzyme-linked immunosorbent assay, immunoblotting, immunoprecipitation, and immunofluorescence. The results demonstrated that the polyclonal antibody against Neuritin had been prepared successfully. The prepared antibody bound to both exogenous and endogenous Neuritin. Importantly, the anti-Neuritin polyclonal antibody could be used in immunoprecipitation assays. CONCLUSIONS: The prepared polyclonal antibody could be used in immunoprecipitation and provide researchers with a useful tool for further investigating the function and mechanism of Neuritin.

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.

Identification of glycosylphosphatidylinositol-anchored proteins and ω-sites using TiO2-based affinity purification followed by hydrogen fluoride treatment.

Glycosylphosphatidylinositol anchored proteins (GPI-APs) in the outer leaflet of the membrane microdomains, commonly referred to as lipid rafts, play important roles in many biological processes such as signal transduction, cell adhesion, protein trafficking, and antigen presentation. From a topological viewpoint, elucidating the presence and localization of GPI-anchor modification sites (ω-sites) is important for the study of the biophysical properties and anchoring mechanisms of these proteins. However, very few reports have actually identified ω-sites of GPI-APs. To enable large-scale site-specific analysis of GPI anchoring, we developed a method for identification of ω-sites by mass spectrometry by combining titanium dioxide-based affinity purification and hydrogen fluoride treatment. This method was able to identify ~3-fold more GPI-APs than our previous method: the new technique identified a total of 73 ω-sites derived from 49 GPI-APs. In 13 of the 49 GPI-APs identified, the GPI-anchor attached to multiple amino acids in the C-terminal site, yielding a variety of different protein species. This method allows us to simultaneously identify many GPI-AP protein species with different ω-sites. We also demonstrated the C-terminal GPI anchor attachment signal peptide, based on information about the GPI anchor binding sites of 49 GPI-APs. Thus, our results provide evidence for new insight into the GPI-anchored proteome and the role of GPI anchoring. BIOLOGICAL SIGNIFICANCE: GPI-anchored proteins (GPI-APs) are localized to the outer leaflet of the plasma membranes. Because the GPI anchor is a complex structure, the identification of GPI-anchored peptides by mass spectrometry has always been considered difficult. To improve the feasibility of large-scale site-specific analysis of GPI anchoring, we developed a method for identification of GPI-anchored peptides by combining titanium dioxide-based affinity purification with hydrogen fluoride treatment. Using this novel technique, we identified a total of 73 ω-sites derived from 49 GPI-APs. These data may help us to develop a comprehensive understanding of the GPI-anchored proteome and the role of GPI anchoring. Moreover, this method could be used to discover GPI-APs as candidate biomarkers.

A Multifaceted Study of Scedosporium boydii Cell Wall Changes during Germination and Identification of GPI-Anchored Proteins.

Scedosporium boydii is a pathogenic filamentous fungus that causes a wide range of human infections, notably respiratory infections in patients with cystic fibrosis. The development of new therapeutic strategies targeting S. boydii necessitates a better understanding of the physiology of this fungus and the identification of new molecular targets. In this work, we studied the conidium-to-germ tube transition using a variety of techniques including scanning and transmission electron microscopy, atomic force microscopy, two-phase partitioning, microelectrophoresis and cationized ferritin labeling, chemical force spectroscopy, lectin labeling, and nanoLC-MS/MS for cell wall GPI-anchored protein analysis. We demonstrated that the cell wall undergoes structural changes with germination accompanied with a lower hydrophobicity, electrostatic charge and binding capacity to cationized ferritin. Changes during germination also included a higher accessibility of some cell wall polysaccharides to lectins and less CH3/CH3 interactions (hydrophobic adhesion forces mainly due to glycoproteins). We also extracted and identified 20 GPI-anchored proteins from the cell wall of S. boydii, among which one was detected only in the conidial wall extract and 12 only in the mycelial wall extract. The identified sequences belonged to protein families involved in virulence in other fungi like Gelp/Gasp, Crhp, Bglp/Bgtp families and a superoxide dismutase. These results highlighted the cell wall remodeling during germination in S. boydii with the identification of a substantial number of cell wall GPI-anchored conidial or hyphal specific proteins, which provides a basis to investigate the role of these molecules in the host-pathogen interaction and fungal virulence.

Mass spectrometric identification of glycosylphosphatidylinositol-anchored peptides.

Glycosylphosphatidylinositol (GPI) anchoring is a post-translational modification widely observed among eukaryotic membrane proteins. GPI anchors are attached to proteins via the carboxy-terminus in the outer leaflet of the cell membrane, where GPI-anchored proteins (GPI-APs) perform important functions as coreceptors and enzymes. Precursors of GPI-APs (Pre-GPI-APs) contain a C-terminal hydrophobic sequence that is involved in cleavage of the signal sequence from the protein and addition of the GPI anchor by the transamidase complex. In order to confirm that a given protein contains a GPI anchor, it is essential to identify the C-terminal peptide containing the GPI-anchor modification site (ω-site). Previously, efficient identification of GPI-anchored C-terminal peptides by mass spectrometry has been difficult, in part because of complex structure of the GPI-anchor moiety. We developed a method to experimentally identify GPI-APs and their ω-sites. In this method, a part of GPI-anchor moieties are removed from GPI-anchored peptides using phosphatidylinositol-specific phospholipase C (PI-PLC) and aqueous hydrogen fluoride (HF), and peptide sequence is then determined by mass spectrometry. Using this method, we successfully identified 10 GPI-APs and 12 ω-sites in the cultured ovarian adenocarcinoma cells, demonstrating that this method is useful for identifying efficiently GPI-APs.

Autoantibodies against exocrine pancreas in Crohn's disease are directed against two antigens: the glycoproteins CUZD1 and GP2.

BACKGROUND: Autoantibodies against exocrine pancreas (PAb) have been reported to be pathognomonic markers of Crohn's disease (CD). Recently, the glycoprotein GP2 has been proposed as the exclusive target for PAb but two equally prevalent binding patterns can be observed in the indirect immunofluorescence test (IIFT) using cryosections of human pancreas: a reticulogranular and a droplet pattern. AIM: To identify autoantigens corresponding to the staining patterns. METHODS: Different lectins were screened for their ability to immobilize PAb-reactive glycoproteins from cell free human pancreas. The glycoproteins were then purified via UEA-I affinity chromatography and identified by mass spectrometry. The two candidate autoantigens were separately expressed in HEK293 cells, and the recombinant cells applied as substrates in IIFT to analyze sera from 96 patients with CD, 89 controls and hybridoma supernatants during the generation of murine monoclonal antibodies. RESULTS: The UEA-I eluate was able to neutralize PAb reactivity of both patterns in IIFT. It contained two major constituents which were identified as the glycoproteins CUZD1 and GP2. With the recombinant cells, 35.4% of the CD patients exhibited positive reactions (CUZD1 alone 19.8%, GP2 alone 9.4%, and both antigens 6.2%). The reaction with the CUZD1 expressing cells was strictly correlated to the reticulogranular pattern, whereas the antibodies causing the droplet pattern stained the GP2 expressing cells. Antigen-capture ELISA using the newly generated monoclonal antibodies against CUZD1 and GP2 verified this relationship. CONCLUSIONS: The concordant reactivities of the different platforms can be regarded as a proof for the authenticity of the two identified autoantigens.

A monoclonal antibody against human UL16-binding protein 3.

The UL16-binding proteins (ULBPs) are a novel family of human MHC class I-related, cell surface proteins that function as ligands for NKG2D. In this study, the gene encoding human ULBP3 was cloned into prokaryotic expression vector pQE30, resulting in a recombinant plasmid pQE30-ULBP3. The pQE30-ULBP3 was transformed into Escherichia coli M15 and induced the expression of recombinant protein ULBP3 (rec-ULBP3). The purified rec-ULBP3 as an antigen was used to immunize BALB/c mice. Through cell fusion, sub-cloning, and screening approach, three hybridoma cell clones expressing monoclonal antibodies (MAb) were acquired. The results from Western blot analysis, flow cytometry, and enzyme-linked immunosorbent assay showed that the hybridoma clones B2-F1-F1 and B4-C5-D11, and not G2-A4-A12, reacted with rec-ULBP3 and nature ULBP3 expressed on the cell surface of the tumor cells. In conclusion, the new MAb described here provides a valuable tool for further investigating ULBP3 function and clinical application.

Strategy for SRM-based verification of biomarker candidates discovered by iTRAQ method in limited breast cancer tissue samples.

Since LC-MS-based quantitative proteomics has become increasingly applied to a wide range of biological applications over the past decade, numerous studies have performed relative and/or absolute abundance determinations across large sets of proteins. In this study, we discovered prognostic biomarker candidates from limited breast cancer tissue samples using discovery-through-verification strategy combining iTRAQ method followed by selected reaction monitoring/multiple reaction monitoring analysis (SRM/MRM). We identified and quantified 5122 proteins with high confidence in 18 patient tissue samples (pooled high-risk (n=9) or low-risk (n=9)). A total of 2480 proteins (48.4%) of them were annotated as membrane proteins, 16.1% were plasma membrane and 6.6% were extracellular space proteins by Gene Ontology analysis. Forty-nine proteins with >2-fold differences in two groups were chosen for further analysis and verified in 16 individual tissue samples (high-risk (n=9) or low-risk (n=7)) using SRM/MRM. Twenty-three proteins were differentially expressed among two groups of which MFAP4 and GP2 were further confirmed by Western blotting in 17 tissue samples (high-risk (n=9) or low-risk (n=8)) and Immunohistochemistry (IHC) in 24 tissue samples (high-risk (n=12) or low-risk (n=12)). These results indicate that the combination of iTRAQ and SRM/MRM proteomics will be a powerful tool for identification and verification of candidate protein biomarkers.

[Prokaryotic expression, purification and antigenicity identification of mouse prostate stem cell antigen].

OBJECTIVE: To amplify mouse prostate stem cell antigen (mPSCA) gene and construct a recombinant plasmid to obtain mPSCA protein and identify its antigenicity. METHODS: The gene of mPSCA was amplified by RT-PCR from mouse prostate cancer cell line RM-1 with the signal peptide sequence removed. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-mPSCA, which was transformed into BL21 (DE3) for mPSCA expression. The fusion protein was purified and identified by SDS-PAGE and Western blotting. The antigenicity of the purified protein was characterized by ELISA. RESULTS: The mPSCA gene was obtained with an identical sequence to that retrieved in GenBank. The prokaryotic expression vector for mPSCA was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated the antigenicity of the purified mPSCA protein. CONCLUSION: The purified mPSCA obtained possesses good antigenicity, which will facilitate further study of immunotherapy for prostate cancer targeting PSCA.

Extragonadal seminoma presenting as a large mass in the pelvic cavity without c-kit-activating mutations.

Extragonadal germ cell tumors are relatively rare tumors, which usually occur in the mediastinum or retroperitoneum. In this report, we present a case of primary seminoma arising in the pelvic cavity. A 58-year-old man with urinary retention and abdominal distension was admitted to our hospital. Computed tomography and magnetic resonance imaging demonstrated a large mass in the pelvic cavity. Histological examination of the specimens obtained by open biopsy revealed seminomatous malignant cells. Immunohistochemical studies detected vimentin, placental alkaline phosphatase and c-kit. Taking these results together with the patient's other clinical manifestations, this case was diagnosed as extragonadal seminoma without c-kit-activating mutations, and chemotherapy followed by radiation therapy was successful. Primary seminoma in the pelvic cavity is extremely rare, but should be considered a cause of pelvic mass formation.

Expression, purification and crystallization of the human UL16-binding protein ULBP1.

UL16-binding proteins (ULBPs) are markers of cellular stress which are upregulated on the surface of virus-infected and tumor cells. Recognition of ULBP1 by the activating receptor NKG2D on the surface of cytotoxic natural killer (NK) and T cells promotes lysis of cells expressing ULBP1 and is an important mechanism of immune surveillance. We report a robust method for the generation of large quantities of crystal-grade recombinant ULBP1 protein. The extracellular portion of human ULBP1 was cloned into a T7 expression vector for expression in Escherichia coli. Unpaired cysteines in the sequence which are predicted not to be involved in the intramolecular disulfide bond formation were mutated to serine. ULBP1 was expressed in E. coli BL21 (DE3) pLysS cells as inclusion bodies. Purified inclusion bodies were solubilized by denaturation in guanidine, and refolded by slow dilution. The refolded protein was purified by size exclusion gel filtration and anion exchange chromatography. Furthermore, we have identified conditions optimal for the crystallization of this protein and have obtained initial diffraction data to 4.6Å from these crystals.