Purification and characterization of recombinant human renin for X-ray crystallization studies

<p>Abstract</p> <p>Background</p> <p>The renin-angiotensin-aldosterone system (RAS) cascade is a major target for the clinical management of hypertension. Although inhibitors of various components of this cascade have been developed successfully, development of renin inhibitors has proven to be problematic. The development of these inhibitors has been hindered by poor bioavailability and complex synthesis. However, despite the challenges of designing renin inhibitors, the enzyme remains a promising target for the development of novel treatments for hypertension. X-ray crystallographic data could greatly assist the design and development of these inhibitors. Here we describe the purification and characterization of recombinant human renin for x-ray crystallization studies.</p> <p>Results</p> <p>A cDNA encoding the full length of native human preprorenin (406 amino acid residues) was introduced into the HEK-293 cell line. A clonal cell line expressing prorenin was generated and grown under serum free conditions in a hollow fiber bioreactor. Prorenin was constitutively secreted and purified directly from the conditioned medium. Concanavalin A chromatography effectively enriched and purified prorenin to 90% homogeneity in a single step. Prorenin was converted to active renin by trypsin digestion to remove the propeptide. Active renin was further purified using a cation exchange column followed by a gel filtration column. Biochemical characterization of the recombinant enzyme showed both binding and catalytic properties were essentially identical to previously reported activities for purified renin. Crystals were grown using this material in our X-ray structure studies, and high resolution diffraction was obtained.</p> <p>Conclusion</p> <p>This present work describes a simple and efficient method for the generation and purification of active human renin. The protein is highly pure and is suitable for supporting structural biology efforts.</p

The MLL-Menin Interaction is a Therapeutic Vulnerability in <em>NUP98</em>-rearranged AML

\ua9 2023 Wolters Kluwer Health. All rights reserved. Chromosomal translocations involving the NUP98 locus are among the most prevalent rearrangements in pediatric acute myeloid leukemia (AML). AML with NUP98 fusions is characterized by high expression of HOXA and MEIS1 genes and is associated with poor clinical outcome. NUP98 fusion proteins are recruited to their target genes by the mixed lineage leukemia (MLL) complex, which involves a direct interaction between MLL and Menin. Here, we show that therapeutic targeting of the Menin-MLL interaction inhibits the propagation of NUP98-rearrranged AML both ex vivo and in vivo. Treatment of primary AML cells with the Menin inhibitor revumenib (SNDX-5613) impairs proliferation and clonogenicity ex vivo in long-term coculture and drives myeloid differentiation. These phenotypic effects are associated with global gene expression changes in primary AML samples that involve the downregulation of many critical NUP98 fusion protein-target genes, such as MEIS1 and CDK6. In addition, Menin inhibition reduces the expression of both wild-type FLT3 and mutated FLT3-ITD, and in combination with FLT3 inhibitor, suppresses patient-derived NUP98-r AML cells in a synergistic manner. Revumenib treatment blocks leukemic engraftment and prevents leukemia-associated death of immunodeficient mice transplanted with NUP98::NSD1 FLT3-ITD-positive patient-derived AML cells. These results demonstrate that NUP98-rearranged AMLs are highly susceptible to inhibition of the MLL-Menin interaction and suggest the inclusion of AML patients harboring NUP98 fusions into the clinical evaluation of Menin inhibitors

The design and characterization of a 300 channel, optimized full-band millimeter filterbank for science with SuperSpec

SuperSpec is an integrated, on-chip spectrometer for millimeter and sub-millimeter astronomy. We report the approach, design optimization, and partial characterization of a 300 channel filterbank covering the 185 to 315 GHz frequency band that targets a resolving power R ~ 310, and fits on a 3.5×5.5 cm chip. SuperSpec uses a lens and broadband antenna to couple radiation into a niobium microstrip that feeds a bank of niobium microstrip half-wave resonators for frequency selectivity. Each half-wave resonator is coupled to the inductor of a titanium nitride lumped-element kinetic inductance detector (LEKID) that detects the incident radiation. The device was designed for use in a demonstration instrument at the Large Millimeter Telescope (LMT)

SuperSpec: On-chip spectrometer design, characterization, and performance

SuperSpec is an integrated, on-chip spectrometer for millimeter and sub-millimeter astronomy. SuperSpec is demonstrating a proof-of-principle multi-beam spectrometer on the sky at the Large Millimeter Telescope (LMT) in Mexico covering the 200 - 300 GHz frequency range with moderate resolution (R ~ 270 - 290). The dual-polarization, three-pixel instrument will consist of 6 SuperSpec spectrometer chips. We present the design and characterization of the devices being used in the first SuperSpec demonstration along with lab testing of the instrument performance

SuperSpec: development towards a full-scale filter bank

SuperSpec is a new spectrometer-on-a-chip technology for submm/mm-wave spectroscopy. SuperSpec stands out from other direct-detection submm spectrometer technologies in that the detectors are coupled to a series of resonant filters along a single microwave feedline instead of using dispersive optics. SuperSpec makes use of kinetic inductance detectors (KIDs) to detect radiation in this filter bank. The small profile of this design makes SuperSpec a natural choice to produce a multi-object spectrometer for tomographic mapping or galaxy redshift surveys. We have recently fabricated a device that is a 50 channel subset of a full 280 channel filter bank, which would cover the 190 - 310 GHz range at R = 275. Analysis of the data from this device informs us of the potential design modifications to enable a high-yield background-limited SuperSpec spectrometer. The results indicate that this subset filter bank can scale up to a full filter bank with only a few collisions in readout space and less than 20% variation in responsivity for the detectors. Additionally, the characterization of this and other prototype devices suggests that the noise performance is limited by generation-recombination noise. Finally, we find that the detectors are sufficiently sensitive for ground-based spectroscopy at R = 100, appropriate for tomographic mapping experiments. Further modifications are required to reach the background limit for R = 400, ideal for spectroscopy of individual galaxies

Full-Array Noise Performance of Deployment-Grade SuperSpec mm-Wave On-Chip Spectrometers

SuperSpec is an on-chip filter bank spectrometer designed for wideband moderate-resolution spectroscopy at millimeter wavelengths, employing TiN kinetic inductance detectors. SuperSpec technology will enable large-format spectroscopic integral field units suitable for high-redshift line intensity mapping and multi-object spectrographs. In previous results, we have demonstrated noise performance in individual detectors suitable for photon noise-limited ground-based observations at excellent mm-wave sites. In these proceedings, we present the noise performance of a full R∼275 spectrometer measured using deployment-ready RF hardware and software. We report typical noise equivalent powers through the full device of ∼3×10⁻¹⁶ W Hz^(−1/2) at expected sky loadings, which are photon noise dominated. Based on these results, we plan to deploy a six-spectrometer demonstration instrument to the Large Millimeter Telescope in early 2020

The design and characterization of a 300 channel, optimized full-band millimeter filterbank for science with SuperSpec

SuperSpec is an integrated, on-chip spectrometer for millimeter and sub-millimeter astronomy. We report the approach, design optimization, and partial characterization of a 300 channel filterbank covering the 185 to 315 GHz frequency band that targets a resolving power R ~ 310, and fits on a 3.5×5.5 cm chip. SuperSpec uses a lens and broadband antenna to couple radiation into a niobium microstrip that feeds a bank of niobium microstrip half-wave resonators for frequency selectivity. Each half-wave resonator is coupled to the inductor of a titanium nitride lumped-element kinetic inductance detector (LEKID) that detects the incident radiation. The device was designed for use in a demonstration instrument at the Large Millimeter Telescope (LMT)