Search

Decidualization is the process of conversion of endometrial stromal cells (ESCs) into decidual stromal cells (DSCs), which is caused by progesterone production that begins during the luteal phase of the menstrual cycle and then increases throughout pregnancy dedicated to support embryonic development. Decidualization deficiency is closely associated with various pregnancy complications, such as recurrent miscarriage (RM). Here, we reported that Src-homology-2-containing phospho-tyrosine phosphatase (SHP2), a key regulator in the signal transduction process downstream of various receptors, plays an indispensable role in decidualization. SHP2 expression was upregulated during decidualization. SHP2 inhibitor RMC-4550 and shRNA mediated SHP2 reduction resulted in a decreased level of phosphorylation of ERK and aberrant cytoplasmic localization of progesterone receptor (PR), coinciding with reduced expression of IGFBP1 and various other target genes of decidualization. Solely inhibiting ERK activity recapitulated these observations. Administration of RMC-4550 led to decidualization deficiency and embryo absorption in mouse. Moreover, reduced expression of SHP2 was detected in decidua of RM patients. Our results revealed that SHP2 is key to PR's nuclear localization, thereby indispensable for decidualization and that reduced expression of SHP2 might be engaged in the pathogenesis of RM.

Platelets are essential components in the tumor microenvironment. For decades, clinical data have demonstrated that cancer patients have a high risk of thrombosis that is associated with adverse prognosis and decreased survival, indicating the involvement of platelets in cancer progression. Increasing evidence confirms that cancer cells are able to induce production and activation of platelets. Once activated, platelets serve as allies of cancer cells in tumor growth and metastasis. They can protect circulating tumor cells (CTCs) against the immune system and detachment-induced apoptosis while facilitating angiogenesis and tumor cell adhesion and invasion. Therefore, antiplatelet agents and platelet-based therapies should be developed for cancer treatment. Here, we discuss the mechanisms underlying the bidirectional cancer-platelet crosstalk and platelet-based therapeutic approaches.

Use of paclitaxel as monotherapy or in combination with other therapeutic agents is a widely employed front-line chemotherapeutic strategy for cervical cancer. However, previous reports have shown that approximately 70% of the patients with cervical cancer develop resistance to paclitaxel. Epithelial-mesenchymal transition (EMT) contributes to the occurrence of chemoresistance in several types of cancer, including cervical cancer. Identification of the critical signaling pathway that regulates the EMT process may provide a novel strategy for avoiding or delaying the emergence of paclitaxel resistance during the treatment of cervical cancer. Herein, we established a paclitaxel-resistant cervical cancer cell line (HeLa-229PTR cells) by culturing parental HeLa-229 cells with increasing concentrations of paclitaxel. We observed elevated expression of Notch1 in HeLa-229PTR cells compared with their parental HeLa-229 cells, indicating its potential involvement in the EMT phenotype of the paclitaxel-resistant cells. Furthermore, silencing of the NOTCH1 gene, as well as treatment with a γ-secretase inhibitor (DAPT) partially reversed the EMT phenotype and significantly enhanced the sensitivity of HeLa-229PTR cells to paclitaxel. Moreover, we found that DAPT could significantly inhibit invasiveness, reduce colony formation activity, and promote apoptosis of HeLa-229PTR cells. Taken together, these results indicated that HeLa-229PTR cells develop the EMT phenotype partly through activation of Notch1 signaling. Thus, inhibition of Notch1 signaling can be a strategy for the reversal of the EMT phenotype and may increase the sensitivity of cervical cancer cells to treatment with paclitaxel.

Receptor tyrosine kinase ROR1 plays an essential role in embryogenesis and is overexpressed in many types of malignant tumors. Studies have demonstrated that it plays an important role in oncogenesis by activating cell survival signaling events, particularly the non-canonical WNT signaling pathway. Antibody-based immunotherapies targeting ROR1 have been developed and evaluated in preclinical and clinical studies with promising outcomes. However, small molecule inhibitors targeting ROR1 are underappreciated because of the initial characterization of ROR1 as a peusdokinase. The function of ROR1 as a tyrosine kinase remains poorly understood, although accumulating evidence have demonstrated its intrinsic tyrosine kinase activity. In this review, we analyzed the structural and functional features of ROR1 and discussed therapeutic strategies targeting this kinase.

Characterization of the dispersion behaviors of spilled liquid hydrogen is necessary from the safety prospective. In this study, the two-phase flow of liquid hydrogen spill is predicted with the mixture multiphase model, Lee model and Realizable k-e model, and the dispersion of hydrogen vapor cloud with the atmospheric inversion layer is numerically analyzed. The inversion layer restrains the upward movement of the cloud and the mixing of the cloud with air, increasing its ground-level hydrogen concentration. The heights of the flammable cloud can be reduced by 5.71%, 10.49% and 12.86%, respectively, with the temperature lapse rates of 0.03, 0.06 and 0.10 K/m in our investigated scenarios. Besides, the restraining effect is strengthened by increasing the ground air temperature if the temperature lapse rate remains unchanged.

Multiple myeloma (MM) is the second most common hematopoietic malignancy and remains an incurable disease. Thus, novel drugs and therapeutic methods are required for patients with MM. The present study aimed to investigate the effect of sirtuin 1 (SIRT1) inhibitor cambinol on the proliferation and apoptosis of myeloma cell lines, RPMI8226 and U266. Moreover, the present study evaluated the underlying molecular mechanisms of proliferation inhibition and apoptosis induced by cambinol. A Cell Counting Kit-8 assay was used to measure the viability of RPMI8226 and U266 cells treated with cambinol. Apoptosis and the cell cycle were analyzed via flow cytometry. The expression levels of caspase-3, poly(ADP-ribose) polymerase 1 (PARP), p53, acetylated p53 (Ac-p53), Bcl-2, cyclin D1 and p21 were detected in cells treated with cambinol using western blot analysis. The results demonstrated that cambinol inhibited the proliferation of RPMI8226 and U266 cells in a time- and dose-dependent manner. Increased apoptosis and G1 cell cycle arrest, together with enhanced procaspase-3 degradation and PARP cleavage were identified in cambinol-treated cells compared with controls. Western blotting results also revealed the upregulation of p53 acetylation and p21, as well as the downregulation of Bcl-2 and cyclin D1 in cells treated with cambinol. In conclusion, the present results suggest that cambinol inhibits the proliferation and induces apoptosis in RPMI8226 and U266 cells by regulating acetylation of p53 via the targeting of SIRT1.

Additional file 1. LC-MS materials, methods and results: S1. Chemicals and Instrumentation; S2. Sample Preparation; S3: LC-MS Analysis method; S4: LC-MS analysis results

Background Acute myeloid leukemia (AML) is a malignant hematological neoplasm of myeloid progenitor cells. Mutations of FLT3 in its tyrosine kinase domain (FLT3-TKD) are found in ~ 8% of patients with AML, with D835Y as the most common substitution. This mutation activates survival signals that drives the disease and is resistant to the first generation FLT3 inhibitors. Development of a highly sensitive method to detect FLT3D835Y is important to direct therapeutic options, predict prognosis, and monitor minimal residual disease in patients with AML. Methods and results In the present study, we developed a highly sensitive FLT3D835Y detection method by using the restriction fragment nested allele-specific PCR technique. The method consists of three steps: 1) initial amplification of DNA samples with PCR primers surrounding the FLT3D835Y mutation site, 2) digestion of the PCR products with restriction enzyme EcoRV that only cleaves the wild type allele, and 3) detection of FLT3D835Y by allele-specific PCR with nested primers. We were able to detect FLT3D835Y with a sensitivity of 0.001% by using purified plasmid DNAs and blood cell DNAs containing known proportions of FLT3D835Y. We analyzed blood cell DNA samples from 64 patients with AML and found six FLT3D835Y-positive cases, two of which could not be detected by conventional DNA sequencing methods. Importantly, the method was able to detect FLT3D835Y in a sample collected from a relapsed patient while the patient was in complete remission with negative MRD determined by flow cytometry. Therefore, our RFN-AS-PCR detected MRD after treatment that was missed by flow cytometry and Sanger DNA sequencing, by conventional methods. Conclusions We have developed a simple and highly sensitive method that will allow for detection of FLT3D835Y at a very low level. This method may have major clinical implications for treatment of AML.

Notch signaling is a highly conserved pathway existed in multicellular organisms. It plays roles in normal human body development, human cancer initiation, progression and metastasis. The Notch negative regulatory region (NRR) is critical for Notch signaling, and cleavage at the S2 site in the NRR ultimately leads to the activation of Notch signaling. To study the function of human NRR1, we expressed the recombinant human NRR1 (rhNRR1) domain in Escherichia coli. After purification, rhNRR1 was obtained with approximately 94% purity according to SDS-PAGE analysis. Furthermore, the polyclonal anti-rhNRR1 serum raised by immunizing mouse with the purified rhNRR1 was able to reduce the generation of active form of Notch1 intracellular domain in HeLa cells, which implied the raised antibody could recognize and bind the natural conformation of Notch1 NRR. Preparation of rhNRR1 by this way is convenient, time-consuming, and could be used to the preparation of anti-NRR1 therapeutic antibody.

Frequently mutated in Acute myeloid leukemia (AML), FLT3 is considered as one of the favorable targets for treatment. The FLT3 internal tandem duplication (ITD) mutation enhances kinase activity and causes hyperactivation of downstream signal transduction. Several small molecule FLT3 inhibitors have developed, but their clinical efficacy is limited due to generation of drug resistance. In this study, we define a new mechanism of drug resistance toward tyrosine kinase inhibitors (TKIs). Initially, we found a rapid decrease in the protein level of tumor suppressor p53 in FLT3-ITD-positive MV4-11 and MOLM13 cells and peripheral blood mononuclear cells (PBMCs) from FLT3-ITD AML patients upon treatment with TKIs including sorafenib, sunitinib and quizartinib. The decrease is not caused by changes in mRNA expression as revealed by qPCR analyses but rather by accelerated protease degradation because the p53 protein was stabilized by proteasome inhibitor MG132. Furthermore, treatment of cells with RG7388, a potent disruptor of p53 and MDM2 interaction, prevented the TKI-induced p53 loss. Since MDM2 is the most important E3 ligase responsible for ubiquitination of p53, the data suggest that TKIs may lead to the degradation of p53 by promoting ubiquitination. Indeed, ubiquitination assays verified that TKIs promoted K48 poly-ubiquitination of p53. Previous studies have demonstrated that activations of FLT3 downstream signaling components such as ERKs and Akt reduce p53 protein stability through ubiquitination by activating MDM2. It is somewhat unexpected that inhibition of FLT3-ITD and its downstream signaling pathways also resulted in decreased p53 stability due to increased ubiquitination. We treated FLT3-ITD-containing cells with specific ERK, AKT and STAT5 inhibitors. Interestingly, while inhibition of ERKs and AKT had no significant effect on the stability of p53, STAT5 inhibition resulted in a reduced level of p53 accompanied by increased K48 poly-ubiquitination. We further analyzed the interaction of p53 with MDM2 in AML cells by using immunoprecipitation. The results showed that the p53-MDM2 interaction was significantly enhanced after treatment with TKIs and STAT5 inhibitors, which was diminished in the presence of RG7388. Subcellular fractionation revealed the presence of p53 and STAT5 in both nucleus and cytoplasm. Treatment of cells with TKIs resulted in a decreased level of p53 and STAT5 in the nucleus, and immunoprecipitation of nuclear proteins with a p53 antibody revealed a reduced association of p53 with STAT5. Taken together, the data suggest that FLT3 inhibitors inhibited nuclear translocation of STAT5 and reduced its interaction of p53 thereby facilitating p53/MDM2 interaction and subsequent ubiquitination and degradation of p53. This study reveals a novel mechanism by which drug resistance to TKIs may occur and further support the use of MDM2/p53 interaction inhibitors in combination with TKIs for treatment of AML. Disclosures No relevant conflicts of interest to declare.

Protein tyrosine phosphatase SHP2 encoded by PTPN11 is a key regulator in growth factor and cytokine signaling. Overwhelming evidence suggests its vital role in hematopoietic stem cell function and hematopoiesis. As a bona fide proto-oncogene product, gain-of-function mutations of SHP2 cause hematological malignancies, most notably juvenile myelomonocytic leukemia (JMML) which bear somatic SHP2 mutations in 35% of cases. Numerous studies have utilized murine models to investigate the role of mutant SHP2 in hematopoiesis and leukemogenesis and successfully produced resembling myeloproliferative neoplasm (MPN) and even full-blown leukemia in recipient animals. However, mutant SHP2-transformed cell lines have not been generated. In the present study, we established oncogenic mutant SHP2-transformed cell lines from erythropoietin (EPO)-dependent HCD-57 erythroid leukemia cells. First, we generated recombinant retroviruses expressing SHP2-D61Y and SHP2-E76K, the two most common SHP2 mutants found in individuals with JMML, by using the pMSCV-IRES-GFP vector. We then infected HCD-57 cells with the recombinant retroviruses. Unlike the parent HCD-57 cells, the infected cells were able to grow in the absence of EPO as demonstrated by viable GFP-positive cells. We further performed semi-solid methylcellulose colony cultures and isolated single clones of EPO-independent HCD57 cells. The isolated clonal cells overexpressed mutant SHP2 and proliferate rapidly in the absence of EPO. In contrast, HCD57 cells infected with retroviruses expressing wild type SHP2 failed to survive in the absence of EPO, indicating only gain-of-function mutant forms of SHP2 have the cell-transformation capability. We also carried out parallel experiments with the pro-B Ba/F3 cell line that require interleukin 3 (IL3) for survival. Interestingly, over-expression of SHP2-D61Y and SHP2-E76K was not sufficient to give rise to IL3-indepdent Ba/F3 cells, suggesting that HCD57 cells have some unique properties making them susceptible for transformation by oncogenic SHP2 mutants. We further performed in vitro and in vivo characterization of transformed HCD57 cells. Cell signaling analyses revealed that both HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells exhibited aberrantly elevated levels of pERK and pAKT in the absence of cytokine stimulation, which was consistent with the notion that gain-of-function SHP2 mutants perturb growth control through deregulation of the Ras signaling pathway. Upon intravenous injection into immunodeficient mice, the SHP2 mutant-transformed HCD57 cells caused acute leukemia with markedly increased spleen. Finally, we screened a small molecule inhibitor library to identify compounds that may specifically target the SHP2 mutants. We found several tyrosine kinase inhibitors including dasatinib and trametinib potently inhibited HCD57-SHP2-D61Y and HCD57-SHP2-E76Kcells but not the parent HCD57 cells. At sub-micromolar concentrations, dasatinib and trametinib abolished elevated ERK and Akt activation caused by the SHP2 mutants. This study not only proves that gain-of function mutations of SHP2 are capable of fully transforming cells but also provides a unique cell system to study pathogenesis of SHP2 mutants and to identify specific inhibitors for drug development. Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a malignant hematopoietic neoplasm featured by impaired differentiation and uncontrolled proliferation of myeloid progenitors. Gain-of-function mutations of FMS-like tyrosine kinase 3 (FLT3) present in 30-40% of patients with AML. In addition, more than 90% of AML blasts aberrantly express FLT3, making FLT3 an attractive therapeutic target for AML. Currently, several small molecule tyrosine kinase inhibitors (TKIs) targeting FLT3 have been approved in the treatment of AML, but they need to be used in combination with chemotherapy because of their limited potency to eliminate leukemic cells as single agents, largely due to the development of secondary inhibitor-resistant FLT3 mutations. Therefore, novel therapeutic strategies targeting FLT3 are needed. In the present study, we developed a FLT3 ligand-emtansine drug conjugate (FL-DM1) that targeted FLT3-positive AML cells with high potency and selectivity. We expressed recombinant human FLT3 ligand (rhFL) in the periplasm of recombinant E. Coli. The protein was purified by a two-step purification system containing Ni-NTA and Phenyl Sepharose. Our purified rhFL was bioactive to stimulate phosphorylation of FLT3 and proliferation of THP-1 cells. Next, we conjugated purified rhFL and emtansine (DM1) with SPDP linker. Reducing and non-reducing SDS-PAGE revealed that rhFL and DM1 were successfully conjugated, evidenced by a band with higher molecular weight of the conjugation product. Previous studies show that DM1 is a drug preferentially targeting proliferating cells by depolymerizing microtubules through binding at the vinca binding site of tubulin. We found that FL-DM1 reserved the physiological function of FLT3 ligand to stimulate proliferation of AML cells by inducing phosphorylation of FLT3 and the downstream signaling protein AKT in immunoblot, potentially enhancing the potency of FL-DM1 to inhibit FLT3-positive AML cells. Furthermore, flow cytometry showed that the surface expression of FLT3 significantly decreased on cells treated by FL-DM1 within two hours, which indicated the internalization of FL-DM1/FLT3 complex on these FLT3-positive AML cells, providing a mechanism of FL-DM1 entering target cells. In cell viability assay, we found that FL-DM1 effectively inhibited FLT3-positive AML cells THP-1 and MV-4-11 with IC50 around 10-30 nM. Also, FL-DM1 induced apoptosis and cell cycle arrest at G2/M phase in these cells detected by flow cytometry. In our previous studies, we generated FLT3-ITD transformed HCD-57 cells. HCD-57 cells are FLT3-negative erythroleukemia cells that depend on erythropoietin for survival. When infected with recombinant retroviruses carrying FLT3-ITD, they acquired ability to proliferate in the absence of EPO. We found that FL-DM1 inhibited HCD-57 cells transformed by FLT3-ITD, but not parental HCD-57 cells without FLT3 expression, indicating the selectivity of FL-DM1 to target FLT3-positive AML cells. In conclusion, our data demonstrated that FL-based drug conjugate can serve as an effective drug to target FLT3-expressing AML cells. Further studies will focus on in-vivo evaluation of FL-DM1 in animal models, the production of uncleavable SMCC linked FL-DM1 with improved in-vivo pharmacokinetic properties, and screening of FL muteins-DM1 conjugates with desired pharmacological properties. Disclosures No relevant conflicts of interest to declare.

B-cell acute lymphoblastic leukemia (B-ALL) is a malignancy of B lymphocytes blocked at an early stage of differentiation, which accounts for approximately 30% of childhood cancers. Almost all B-ALL blasts aberrantly express FMS-like tyrosine kinase 3 (FLT3), a receptor tyrosine kinase involved in the development of dendritic cells and B lymphocytes. In B-ALL, the high expression of FLT3 in blasts significantly increases the odds of relapse/death in patients, indicating a pathological role of FLT3 in B-ALL. Note that gain-of-function mutations of FLT3 are relatively rare ( Disclosures No relevant conflicts of interest to declare.

Acute myeloid leukemia (AML) is a hematological malignancy featured by impaired differentiation and uncontrolled proliferation of myeloid blasts. FLT3 internal tandem duplication (ITD) presents in 30-40% patients with AML, which serves as an independent poor prognostic marker and an attractive therapeutic target. Up to date, several tyrosine kinase inhibitors (TKIs) targeting FLT3 have been approved by FDA in the treatment of AML. However, FLT3 TKIs as single agents have limited effects to eliminate AML cells due to multiple drug-resistant mechanisms, including secondary FLT3 mutations, alternatively activated cell survival pathways, upregulation of FLT3 ligand, and downregulation of tumor suppressor genes. In the present study, we found that FLT3 TKIs decreased tumor suppressor p53 protein level by downregulation of YOD1 through miR-181 in FLT3-ITD mutant AML cells. In our previous studies, we generated FLT3-ITD transformed HCD-57 cells. HCD-57 cells are erythroleukemia cells that depend on erythropoietin for survival. When infected with recombinant retroviruses carrying FLT3-ITD, they acquired ability to proliferate in the absence of EPO. By using transcriptome analysis with RNAseq, we identified multiple differentially expressed miRNAs in HCD-57 transformed by FLT3-ITD, compared with parental HCD-57 cells. miR-181a-5p and miR-181b-5p were among these highly differentially expressed miRNAs. These two miRNAs were predicted to bind in 3'UTR of deubiquitinase YOD1 by using TargetScanHuman 7.2, an online tool to predict biological targets of miRNAs. Previous studies have shown that the ubiquitination and protein level of p53 is dysregulated due to overexpressed E3 ligase MDM2/4 in AML cells, but few studies focused on deubiquitinase of p53. We found that deubiquitinase YOD1 interacted with p53 by immunoprecipitation. Overexpression of YOD1 prevented degradation of p53 led by cycloheximide, a protein synthesis inhibitor. We further found that overexpression of YOD1 resulted in decreased ubiquitination of p53, indicating that YOD1 stabilized p53 protein through deubiquitination. Subsequently, we detected the expression of miR-181, YOD1 and p53 in a FLT3-ITD positive AML cell line MV-4-11 treated by FLT3 TKIs sorafenib, sunitinib and quizartinib. Real-time quantitative PCR showed that the treatment of FLT3 TKIs upregulated the expression of miR-181a-5p/miR-181b-5p, and downregulated mRNA level of their predicted target YOD1. The mRNA level of p53 remained unchanged but its protein level decreased with enhanced ubiquitination in MV-4-11 cells treated by FLT3 TKIs in the ubiquitination assay. These data suggested that FLT3 TKIs could reduce the stability of p53 by regulating miRNA-targeted YOD1. In addition, we collected peripheral blood mononuclear cells (PBMCs) from patients with AML and age-matched healthy donors. We found increased expression of miR-181a-5p and miR-181b-5p and decreased expression of YOD1 in PBMCs from AML patients compared with healthy donors, suggesting a pathological role of miR-181 to regulate YOD1/53 pathway in AML. In conclusion, our data showed that FLT3 TKIs induced instability of p53 by miR-181 mediated downregulation of YOD1. YOD1 as a novel deubiquitinase of p53 could play important roles in drug-resistance and progression of AML. Disclosures No relevant conflicts of interest to declare.

BACKGROUND Moringa leaf is licensed as a new food ingredient and is rich in nutrients. However, due to its high cellulose content, the nutrients are difficult to digest and absorb, which limits the product development and application of Moringa leaf. RESULTS We chose four bacterial strains—Trichoderma reesei CICC 2626, Bacillus pumilus CICC 10440, Bacillus subtilis subsp. CICC 21934, and Bacillus sp. CICC 21942—to ferment with Moringa leaf powder. The results of solid fermentation and liquid fermentation show that the solid-state fermentation using Bacillus pumilus CICC 10440 in a 1:60 ratio with Moringa leaf powder produced the greatest amount of soluble protein (285 mg/g, 2.42 times higher than the control) in 24 h. Using a cellulase activity assay and western blotting, we found that Bacillus pumilus CICC 10440 uses endoglucanase to dissolve the cellulose to release nutrients from the Moringa oleifera leaf. We expanded the fermentation scale of the Moringa oleifera leaf in a 200 l fermenter and produced a 50-kg batch of Moringa oleifera product. CONCLUSION The results showing improvement in the nutrient release of Moringa leaf via microbial fermentation are a promising start and provide a basis for the further product development of Moringa leaf.

Major histocompatibility complex (MHC) class I-related chain A/B (MICA/B) is a type of stress-induced molecule that plays an important role in tumor surveillance. MICA/B shares a similar structure with MHC class I molecules, but MICA/B contains a closed cleft, not an open one, in its N-terminal alpha1 domain. The alpha 1 domain was believed to have no roles in antigen presentation, because the closed cleft provides limited space for binding with known molecules, and the cleft of MICA/B have been reported no known functions. To study the possible function of the cleft located in human MICA/B's alpha 1 domain, we attempted to express the human MICB-α1 (hMICB-α1) domain allele protein, which is approximately 20.5 kDa, by utilizing an Escherichia coli (E. coli) secretory pathway. Protein expression was accomplished through the phosphate-limited inducible promoter. After purification using ammonium sulfate precipitation, phenyl hydrophobic Sepharose, SP Sepharose and HisTrap affinity Sepharose, recombinant human MICB-α1 (rhMICB-α1) was obtained with 94.3% purity. The binding capacity of rhMICB-α1 with natural killer group 2, member D (NKG2D) was evaluated in vitro. The results demonstrated that rhMICB-α1 can be prepared through the E. coli secretory pathway. Purified rhMICB-α1 protein was able to functionally bind with NKG2D. This method can be further used to obtain functionally active rhMICB-α1 protein, which can served as the basis for further studies of the possible function of the MICB cleft.

Notch signaling is a highly conserved pathway existed in multicellular organisms. It plays roles in normal human body development, human cancer initiation, progression and metastasis. The Notch negative regulatory region (NRR) is critical for Notch signaling, and cleavage at the S2 site in the NRR ultimately leads to the activation of Notch signaling. To study the function of human NRR1, we expressed the recombinant human NRR1 (rhNRR1) domain in Escherichia coli. After purification, rhNRR1 was obtained with approximately 94% purity according to SDS-PAGE analysis. Furthermore, the polyclonal anti-rhNRR1 serum raised by immunizing mouse with the purified rhNRR1 was able to reduce the generation of active form of Notch1 intracellular domain in HeLa cells, which implied the raised antibody could recognize and bind the natural conformation of Notch1 NRR. Preparation of rhNRR1 by this way is convenient, time-consuming, and could be used to the preparation of anti-NRR1 therapeutic antibody.

In the fields of drug discovery and protein science, small quantities of proteins are always needed to investigate or validate protein–protein (or protein–small molecule) interactions. Traditional transient or stable expression method to obtain recombinant proteins in eukaryotic systems can be laborious and time-consuming, especially when multiple protein variants are required. Here, we present a fast and convenient method for obtaining small quantities of recombinant human epidermal growth factor receptor (rhEGFR) ectodomain protein, which could be efficiently extended to the expression of other eukaryotic proteins. Human EGFR ectodomain gene was inserted into the plasmid pBMN-GFP and recombinant plasmid was transfected into HEK 293 T cells. In the presence of hygromycin, cells with the integrated human EGFR ectodomain gene were selected and proliferated. rhEGFR ectodomain in cell culture supernatant was purified using serial connected diethylaminoethyl Sepharose column and Ni-NTA Sepharose column. Purity of the final purified rhEGFR ectodomain was over 95% according to SDS-PAGE analysis. Moreover, the purified target protein was biological active via measuring the affinity between the rhEGFR ectodomain and rhEGF. Our method could greatly facilitate research in the areas of protein science, protein structural biology, and drug discovery.

To prepare the monoclonal antibody (mAb) against recombinant human interferon-β1a (rhIFN-β1a).BALB/c mice were immunized with purified rhIFN-β1a. The spleen lymphocyte cells from the one whose blood specific antibody titer exceeded 1:10 000 were fused with Sp2/0 cells. The fused cells went through HAT selection, serial dilution, and positive selection until stable hybridoma cell lines were obtained. The equilibrium dissociation constant (K(D)) of antibodies was calculated based on ELISA result. The cell line with the best KD value was cultured in a stirred-tank bioreactor with a fed-batch strategy to produce secreted anti-rhIFN-β1a monoclonal antibody. The mAb released into the cell culture supernatant were purified by ultrafiltration and protein-G affinity chromatography.Thirteen hybridoma cell lines which stably produced anti-rhIFN-β1a antibody were retrieved from standard hybridoma fusion and selection procedures. One of the 13 cell lines, 1E8 with the KD value of 4.6 × 10⁻⁹ mol/L was cultured and antibody titer in the cell culture supernatant reached 1:5000. The mAb with 90% purity was recovered from the two-steps purification.The anti-rhIFN-β1a mAb with high purity has been successfully obtained.