Large-scale preparation of active caspase-3 in E. coli by designing its thrombin-activatable precursors

<p>Abstract</p> <p>Background</p> <p>Caspase-3, a principal apoptotic effector that cleaves the majority of cellular substrates, is an important medicinal target for the treatment of cancers and neurodegenerative diseases. Large amounts of the protein are required for drug discovery research. However, previous efforts to express the full-length caspase-3 gene in <it>E. coli </it>have been unsuccessful.</p> <p>Results</p> <p>Overproducers of thrombin-activatable full-length caspase-3 precursors were prepared by engineering the auto-activation sites of caspase-3 precursor into a sequence susceptible to thrombin hydrolysis. The engineered precursors were highly expressed as soluble proteins in <it>E. coli </it>and easily purified by affinity chromatography, to levels of 10–15 mg from 1 L of <it>E. coli </it>culture, and readily activated by thrombin digestion. Kinetic evaluation disclosed that thrombin digestion enhanced catalytic activity (<it>k</it>_cat/<it>K</it>_<it>M</it>) of the precursor proteins by two orders of magnitude.</p> <p>Conclusion</p> <p>A novel method for a large-scale preparation of active caspase-3 was developed by a strategic engineering to lack auto-activation during expression with amino acid sequences susceptible to thrombin, facilitating high-level expression in <it>E. coli</it>. The precursor protein was easily purified and activated through specific cleavage at the engineered sites by thrombin, generating active caspase-3 in high yields.</p

Developmental regulation of the immunoglobulin kappa 3\u27 enhancer: Characterization of negative regulatory elements and cloning of a transcriptional activator/repressor, NF -E1

The immunoglobulin kappa 3\sp\prime enhancer ($\kappa$E3\sp\prime) activity is developmentally regulated during B-cell development. It is inactive at the pre-B cell stage but becomes active at the B-cell and plasma cell stages. The mechanism underlying the silencing of the $\kappa$E3\sp\prime enhancer at the pre-B cell stage is unclear. This study was initiated to identify cis- and trans-acting factors that are involved in the developmental regulation of $\kappa$E3\sp\prime enhancer activity. Transfection of $\kappa$E3\sp\prime enhancer deletion constructs into pre-B cells showed that the 132 bp core of the enhancer is active at this stage, but it is repressed by multiple negative-acting flanking DNA sequences. Deletion of either of these flanking sequences results in activation of the $\kappa$E3\sp\prime enhancer in pre-B cells. A nuclear factor, NF-E1, that binds to negative-acting DNA sequences on the 3\sp\prime side of the enhancer core was identified and is identical to the protein that binds to the $\mu$E1 site of the immunoglobulin heavy chain enhancer. Human cDNA clones encoding NF-E1 were isolated by oligonucleotide screening of a $\lambda$gt11 expression library prepared from human B-cells. Analysis of the predicted amino acid sequence revealed that NF-E1 is a zinc finger protein which belongs to the Kruppel family and contains amino acid sequences common to both transcriptional activators and transcriptional repressors. Electrophoretic mobility shift assays with NF-E1 protein prepared in E. coli indicate that eukaryotic specific protein modifications are not necessary for sequence specific DNA binding activity, but the intact zinc finger domain of the NF-E1 protein is necessary for its DNA binding. The presence of NF-E1 binding sites in both negative and positive regulatory elements suggests that it may have a dual function. Consistent with this expectation, cotransfection experiments showed that NF-E1 functions as an activator as well as a repressor in a dose-dependent manner. Dissection of the NF-E1 protein showed that the region of the protein responsible for repression lies near the carboxy terminus encompassing the zinc finger domain and is distinct from the region necessary for activation. Identification of multiple negative regulatory elements and isolation of NF-E1 have furthered our understanding of how the $\kappa$E3\sp\prime enhancer may be regulated during B-cell development, and how a single transcription factor can result in either transcriptional activation or repression

A novel cervical cancer suppressor 3 (CCS-3) interacts with the BTB domain of PLZF and inhibits the cell growth by inducing apoptosis

AbstractPromyelocytic leukemia zinc finger protein (PLZF) is a sequence-specific, DNA binding, transcriptional repressor differentially expressed during embryogenesis and in adult tissues. PLZF is known to be a negative regulator of cell cycle progression. We used PLZF as bait in a yeast two-hybrid screen with a cDNA library from the human ovary tissue. A novel cervical cancer suppressor 3 (CCS-3) was identified as a PLZF interacting partner. Further characterization revealed the BTB domain as an interacting domain of PLZF. Interaction of CCS-3 with PLZF in mammalian cells was also confirmed by co-immunoprecipitation and in vitro binding assays. It was found that, although CCS-3 shares similar homology with eEF1A, the study determined CCS-3 to be an isoform. CCS-3 was observed to be downregulated in human cervical cell lines as well as in cervical tumors when compared to those from normal tissues. Overexpression of CCS-3 in human cervical cell lines inhibits cell growth by inducing apoptosis and suppressing human cyclin A2 promoter activity. These combined results suggest that the potential tumor suppressor activity of CCS-3 may be mediated by its interaction with PLZF

Integrated microfluidic preconcentration and nucleic amplification system for detection of influenza a virus H1N1 in saliva

InfluenzaAviruses are often present in environmental and clinical samples at concentrations below the limit of detection (LOD) of molecular diagnostics. Here we report an integrated microfluidic preconcentration and nucleic amplification system (μFPNAS) which enables both preconcentration of influenza A virus H1N1 (H1N1) and amplification of its viral RNA, thereby lowering LOD for H1N1. H1N1 virus particles were first magnetically preconcentrated using magnetic nanoparticles conjugated with an antibody specific for the virus. Their isolated RNA was amplified to cDNA through thermocycling in a trapezoidal chamber of the μFPNAS. A detection limit as low as 100 TCID50 (50% tissue culture infective dose) in saliva can be obtained within 2 hours. These results suggest that the LOD of molecular diagnostics for virus can be lowered by systematically combining immunomagnetic separation and reverse transcriptase-polymerase chain reaction (RT-PCR) in one microfluidic device

Stem Cell Factor SOX9 Interacts with a Cell Death Regulator RIPK1 and Results in Escape of Cancer Stem Cell Death

High-grade ovarian cancer (HGOC) is the most lethal gynecological cancer, with high metastasis and recurrence. Cancer stem cells (CSCs) are responsible for its apoptosis resistance, cancer metastasis, and recurrence. Thus, targeting CSCs would be a promising strategy for overcoming chemotherapy resistance and improving patient prognosis in HGOC. Among upregulated oncogenic proteins in HGOC, we found that transcription factor SOX9 showed a strong correlation with stemness-regulating ALDH1A1 and was localized predominantly in the cytoplasm of HGOC with lymph node metastasis. In order to address the role of unusual cytoplasmic SOX9 and to explore its underlying mechanism in HGOC malignancy, a Y2H assay was used to identify a necroptotic cell death-associated cytoplasmic protein, receptor-interacting serine/threonine protein kinase 1 (RIPK1), as a novel SOX9-interacting partner and further mapped their respective interacting domains. The C-terminal region containing the transactivation domain of SOX9 interacted with the death domain of R1PK1. Consistent with its stemness-promoting function, SOX9 knockdown in vitro resulted in changes in cell morphology, cell cycle, stem cell marker expression, cell invasion, and sphere formation. Furthermore, in vivo knockdown completely inhibited tumor growth in mouse xenograft model. We propose that cytoplasmic SOX9-mediated cell death suppression would contribute to cancer stem cell survival in HGOC