Characterization of CprK1, a CRP/FNR-Type Transcriptional Regulator of Halorespiration from Desulfitobacterium hafniense

The recently identified CprK branch of the CRP (cyclic AMP receptor protein)-FNR (fumarate and nitrate reduction regulator) family of transcriptional regulators includes proteins that activate the transcription of genes encoding proteins involved in reductive dehalogenation of chlorinated aromatic compounds. Here we report the characterization of the CprK1 protein from Desulfitobacterium hafniense, an anaerobic low-G+C gram-positive bacterium that is capable of reductive dechlorination of 3-chloro-4-hydroxyphenylacetic acid (Cl-OHPA). The gene encoding CprK1 was cloned and functionally overexpressed in Escherichia coli, and the protein was subsequently purified to homogeneity. To investigate the interaction of CprK1 with three of its predicted binding sequences (dehaloboxes), we performed in vitro DNA-binding assays (electrophoretic mobility shift assays) as well as in vivo promoter probe assays. Our results show that CprK1 binds its target dehaloboxes with high affinity (dissociation constant, 90 nM) in the presence of Cl-OHPA and that transcriptional initiation by CprK1 is influenced by deviations in the dehaloboxes from the consensus TTAAT----ATTAA sequence. A mutant CprK1 protein was created by a Val→Glu substitution at a conserved position in the recognition α-helix that gained FNR-type DNA-binding specificity, recognizing the TTGAT----ATCAA sequence (FNR box) instead of the dehaloboxes. CprK1 was subject to oxidative inactivation in vitro, most likely caused by the formation of an intermolecular disulfide bridge between Cys11 and Cys200. The possibility of redox regulation of CprK1 by a thiol-disulfide exchange reaction was investigated by using two Cys→Ser mutants. Our results indicate that a Cys11-Cys200 disulfide bridge does not appear to play a physiological role in the regulation of CprK1

Transgenic pigs expressing near infrared fluorescent protein—A novel tool for noninvasive imaging of islet xenotransplants

Background Islet xenotransplantation is a promising concept for beta-cell replacement therapy. Reporter genes for noninvasive monitoring of islet engraftment, graft mass changes, long-term survival, and graft failure support the optimization of transplantation strategies. Near-infrared fluorescent protein (iRFP) is ideal for fluorescence imaging (FI) in tissue, but also for multispectral optoacoustic tomography (MSOT) with an even higher imaging depth. Therefore, we generated reporter pigs ubiquitously expressing iRFP. Methods CAG-iRPF720 transgenic reporter pigs were generated by somatic cell nuclear transfer from FACS-selected stable transfected donor cells. Neonatal pig islets (NPIs) were transplanted into streptozotocin-diabetic immunodeficient NOD-scid IL2Rgnull (NSG) mice. FI and MSOT were performed to visualize different numbers of NPIs and to evaluate associations between signal intensity and glycemia. MSOT was also tested in a large animal model. Results CAG-iRFP transgenic NPIs were functionally equivalent with wild-type NPIs. Four weeks after transplantation under the kidney capsule, FI revealed a twofold higher signal for 4000-NPI compared to 1000-NPI grafts. Ten weeks after transplantation, the fluorescence intensity of the 4000-NPI graft was inversely correlated with glycemia. After intramuscular transplantation into diabetic NSG mice, MSOT revealed clear dose-dependent signals for grafts of 750, 1500, and 3000 NPIs. Dose-dependent MSOT signals were also revealed in a pig model, with stronger signals after subcutaneous (depth ∼6 mm) than after submuscular (depth ∼15 mm) placement of the NPIs. Conclusions Islets from CAG-iRFP transgenic pigs are fully functional and accessible to long-term monitoring by state-of-the-art imaging modalities. The novel reporter pigs will support the development and preclinical testing of novel matrices and engraftment strategies for porcine xeno-islets