Expression Of Mammalian Protein Research Articles

Expression and Purification of Mammalian NMDA Receptor Protein for Functional Characterization.

N-methyl-D-aspartate (NMDA) receptors are critical for brain function and serve as drug targets for the treatment of neurological and psychiatric disorders. They typically form thetetrameric assembly of GluN1-GluN2 (2A to 2D) subtypes, with their diverse three-dimensional conformations linked with the physiologically relevant function in vivo. Purified proteins of tetrameric assembled NMDA receptors have broad applications in the structural elucidation, hybridoma technology for antibody production, and high-throughput drug screening. However, obtaining sufficient quantity and monodisperse NMDA receptor protein is still technically challenging. Here, we summarize a paradigm for the expression and purification of diverse NMDA receptor subtypes, with detailed descriptions on screening constructs by fluorescence size-exclusion chromatography (FSEC), generation of recombinant baculovirus, expression in theeukaryotic expression system, protein purification by affinity chromatography and size-exclusion chromatography (SEC), biochemical and functional validation assays.

SLM2 Is A Novel Cardiac Splicing Factor Involved in Heart Failure due to Dilated Cardiomyopathy

Alternative mRNA splicing is a fundamental process to increase the versatility of the genome. In humans, cardiac mRNA splicing is involved in the pathophysiology of heart failure. Mutations in the splicing factor RNA binding motif protein 20 (RBM20) cause severe forms of cardiomyopathy. To identify novel cardiomyopathy-associated splicing factors, RNA-seq and tissue-enrichment analyses were performed, which identified up-regulated expression of Sam68-Like mammalian protein 2 (SLM2) in the left ventricle of dilated cardiomyopathy (DCM) patients. In the human heart, SLM2 binds to important transcripts of sarcomere constituents, such as those encoding myosin light chain 2 (MYL2), troponin I3 (TNNI3), troponin T2 (TNNT2), tropomyosin 1/2 (TPM1/2), and titin (TTN). Mechanistically, SLM2 mediates intron retention, prevents exon exclusion, and thereby mediates alternative splicing of the mRNA regions encoding the variable proline-, glutamate-, valine-, and lysine-rich (PEVK) domain and another part of the I-band region of titin. In summary, SLM2 is a novel cardiac splicing regulator with essential functions for maintaining cardiomyocyte integrity by binding to and processing the mRNAs of essential cardiac constituents such as titin.

Effect of Dietary Inulin Supplementation on Growth Performance, Carcass Traits, and Meat Quality in Growing-Finishing Pigs.

Simple SummaryDietary fiber has attracted considerable research interest worldwide. Inulin is a critical soluble dietary fiber. This study investigated the effects of dietary inulin supplementation on the growth performance and meat quality in pigs, which provided novel insights into the application of inulin for the livestock industry.Inulin is one of the commercially feasible dietary fibers that has been implicated in regulating the gut health and metabolism of animals. This experiment was conducted to investigate the effect of dietary inulin supplementation on growth performance and meat quality in growing–finishing pigs. Thirty-six Duroc × Landrace × Yorkshire White growing barrows (22.0 ± 1.0 kg) were randomly allocated to two dietary treatments consisting of a basal control diet (CON) or basal diet supplemented with 0.5% inulin (INU). Results showed that inulin supplementation tended to increase the average daily gain (ADG) at the fattening stage (0.05 < p < 0.10). Inulin significantly increased the dressing percentage (p < 0.05) and tended to increase the loin-eye area. The serum concentrations of insulin and IGF-I were significantly higher (p < 0.05) in the INU group than in the CON group. Moreover, inulin supplementation significantly elevated the expression level of myosin heavy chain II b (MyHC IIb) in the longissimus dorsi (p < 0.05). Inulin significantly upregulated the expression of mammalian rapamycin target protein (mTOR) but decreased (p < 0.05) the expression level of muscle-specific ubiquitin ligase MuRF-1. These results show the beneficial effect of inulin supplementation on the growth performance and carcass traits in growing–finishing pigs, and will also facilitate the application of inulin in swine production.

Purification and Fibrillation of Full-Length Recombinant PrP.

Misfolding and aggregation of prion protein are related to several neurodegenerative diseases in humans such as Creutzfeldt-Jakob disease, fatal familial insomnia, and Gerstmann-Straussler-Scheinker disease. A growing number of applications in the prion field including assays for detection of PrPSc and methods for production of PrPSc de novo require recombinant prion protein (PrP) of high purity and quality. Here, we report an experimental procedure for expression and purification of full-length mammalian prion protein. This protocol has been proved to yield PrP of extremely high purity that lacks PrP adducts, oxidative modifications, or truncation, which is typically generated as a result of spontaneous oxidation or degradation. We also describe methods for preparation of amyloid fibrils from recombinant PrP in vitro. Recombinant PrP fibrils can be used as a noninfectious synthetic surrogate of PrPSc for development of prion diagnostics including generation of PrPSc-specific antibody.

3. Gene Delivery and Expression Series Overexpression of Recombinant Protein in Bacterial Cells (2)

The protein expression system in bacteria is widely used to overproduce recombinant proteins. Many established expression vectors are on the market. The Escherichia coli expression system is extremely useful for biochemical and biophysical analysis. Here, we describe the results of the expression of mammalian protein in E. coli and an experiment on the structural analysis of recombinant protein.

Intracellular localisation and expression of mammalian CDC2 protein during myogenic differentiation

Abstract . Myogenic differentiation involves withdrawal of myoblasts from the cell cycle and fusion to form multinucleate myotubes. To examine the role that cell cycle control genes may play in this process, we investigated the steady state levels of CDC2 protein and RNA during myogenesis of L6E9 rat myoblasts. Indirect immunofluorescence using a CDC2 affinity-purified antibody showed that this protein is localised exclusively in the cytoplasm with a higher concentration perinuclearly. Both protein and RNA levels were down-regulated to similar extents early in the differentiation process, as cells became quiescent. There was a further down-regulation of protein after fusion to form myotubes. Autonomous expression of CDC2 protein in L6E9 cells, after stable transfection with a metallothionein: CDC2 gene construct, failed to inhibit the differentiation process. This suggests that, although there is down-regulation in levels of CDC2 RNA and protein during myogenesis, this phenomenon per se does not play a primary role in controlling the differentiation process. If CDC2 is involved in control of differentiation, this must depend on post-translational modification of the protein.