We present an example of expression and purification of a biologically active G protein coupled receptor (GPCR) from yeast. An expression vector was constructed to encode the Saccharomyces cerevisiae GPCR α-factor receptor (Ste2p, the STE2 gene product) containing a 9-amino acid sequence of rhodopsin that served as an epitope/affinity tag. In the construct, two glycosylation sites and two cysteine residues were removed to aid future structural and functional studies. The receptor was expressed in yeast cells and was detected as a single band in a western blot indicating the absence of glycosylation. Ligand binding and signaling assays of the epitope-tagged, mutated receptor showed it maintained the full wild-type biological activity. For extraction of Ste2p, yeast membranes were solubilized with 0.5 % n-dodecyl maltoside (DM). Approximately 120 μg of purified α-factor receptor was obtained per liter of culture by single-step affinity chromatography using a monoclonal antibody to the rhodopsin epitope. The binding affinity (Kd) of the purified α-factor receptor in DM micelles was 28 nM as compared to Kd = 12.7 nM for Ste2p in cell membranes, and approximately 40 % of the purified receptor was correctly folded as judged by ligand saturation binding. About 50 % of the receptor sequence was retrieved from MALDI-TOF and nanospray mass spectrometry after CNBr digestion of the purified receptor. The methods described will enable structural studies of the α-factor receptor and may provide an efficient technique to purify other GPCRs that have been functionally expressed in yeast.

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The aim of this study is to evaluate the performance of the GHP system with 45 cast-in-place energy piles(CEP) for a commercial building. In order to demonstrate the feasibility of a sustainable performance of the system, transient simulations were conducted over 1-year and 20-year periods, respectively. The 1-year simulation results showed that the maximum and minimum temperatures of brine returning from the CEPs were 23.91℃ and 6.66℃, which were in a range of design target temperatures. In addition, after 20 years' operation, these returning temperatures decreased to 21.24℃ and 3.68℃, and finally reached to stable state. Annual average extraction heat of cast-in-place energy piles was 94.3 MWh and injection heat was 65.7 MWh from the 20 years of simulation results. Finally, it is expected this GHP system can operate with average heating SPF of more than 3.45 for long-term.

The CRISPR/Cas9 system is a robust, efficient, and cost-effective gene editing tool widely adopted in translational studies of ocular diseases. However, in vivo CRISPR-based editing in animal models poses challenges such as the efficient delivery of the CRISPR components in viral vectors with limited packaging capacity and a Cas9-associated immune response. Using a germline Cas9-expressing mouse model would help to overcome these limitations. Here, we evaluated the long-term effects of SpCas9 expression on retinal morphology and function using Rosa26-Cas9 knock-in mice. We observed abundant SpCas9 expression in the RPE and retina of Rosa26-Cas9 mice using the real-time polymerase chain reaction (RT-PCR), Western blotting, and immunostaining. SD-OCT imaging and histological analysis of the RPE, retinal layers, and vasculature showed no apparent structural abnormalities in adult and aged Cas9 mice. Full-field electroretinogram of adult and aged Cas9 mice showed no long-term functional changes in the retinal tissues because of constitutive Cas9 expression. The current study showed that both the retina and RPE maintain their phenotypic and functional features in Cas9 knock-in mice, establishing this as an ideal animal model for developing therapeutics for retinal diseases.