Pulldown assay is an in vitro method for studies of protein-protein interactions, in which tagged proteins are usually expressed as the bait to enrich other proteins that could bind to them. In this technology, the GST tag is broadest used for its modest size and hydrophilic property. In most cases, the GST tag could increase the hydrophility of the fusion protein and help to avoid the formation of inclusion bodies. However, in the other few cases, the target protein may be strongly hydrophobic or have complicated structures that were hard to fold and assemble in correct conformations without champerons, and even the existence of GST tag could not make them soluble. These proteins were always expressed as inclusion bodies and had no functions. LMO2 was a small molecular weight and insoluble protein, in this study, GST system and MBP system were used to express GST-LMO2 and MBP-LMO2 fusion proteins, respectively. We found that GST-LMO2 fusion protein was expressed as inclusion bodies whereas MBP-LMO2 fusion protein was expressed in soluble form. Moreover, the production rate of MBP-LMO2 was also much higher than GST-LMO2. Then MBP-LMO2 fusion proteins and renatured GST-LMO2 fusion proteins were used as bait in pulldown assay to study the interaction between LMO2 and endogenous GATA1 in K562 cells. Western blot analyses showed that both of these proteins could bind to endogenous GATA1 in K562 cells, but recovered GATA1 protein by MBP-LMO2 fusion protein was much more than GST-LMO2 fusion protein. These results suggest that using of MBP system is a helpful attempt in the case of studying small molecular weight, strong hydrophobic proteins.
Adaptor Proteins, Signal Transducing ; Carrier Proteins ; chemistry ; Chemical Precipitation ; DNA-Binding Proteins ; chemistry ; GATA1 Transcription Factor ; chemistry ; Genetic Vectors ; Glutathione Transferase ; chemistry ; Humans ; K562 Cells ; LIM Domain Proteins ; Maltose-Binding Proteins ; Metalloproteins ; chemistry ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Renaturation ; Proto-Oncogene Proteins ; chemistry ; Recombinant Fusion Proteins ; genetics ; metabolism

Bone morphogenetic protein 2 (BMP2), which belongs to the transforming growth factor-beta (TGF-beta) superfamily, is a multifunctional molecule with distinct abilities to induce bone formation. BMP2 has been identified to have eminent pharmaceutical importance for clinical application. We previously constructed stable cell line in Chinese hamster ovary cells (CHO) that highly expressed recombinant human BMP2 (rhBMP2). For large-scale production of the recombinant protein used in clinical application, it is critical to have both high expression and stability of the protein. In the present study, the stability of the cell line (rCHO(hBMP2)-C8) with the highest expression, as well as the stability of rhBMP2 protein were investigated systematically. We cultured the rCHO (hBMP2)-C8 cell line in the presence or absence of MTX for two months, the cell growth and rhBMP2 production characteristics were examined during the culture; we found the duration that the rCHO(hBMP2)-C8 cell line could secret rhBMP2 continually into the serum-free medium. Moreover, we detected the temperature sensitivity of rhBMP2 in culture medium. This study will contribute to our understanding for further producing rhBMP2 by large-scale culture technology.
Animals ; Bone Morphogenetic Protein 2 ; biosynthesis ; genetics ; CHO Cells ; Cell Culture Techniques ; methods ; Cricetinae ; Cricetulus ; Culture Media ; Genetic Vectors ; genetics ; Humans ; Recombinant Proteins ; biosynthesis ; genetics

Objective:The aim of this study was to evaluate the safety and efficacy of prostatistic urethral lift (PUL) in treating benign prostate hyperplasia(BPH) through systematic review and Meta-analysis.Methods:A systematic literature search on CNKI, Wanfang, VIP, PubMed, Web of Science, Cochrane Library and Chinese Clinical Trial Registry to identify the relevant studies and data before September 2021. Information was extracted from each eligible article. All statistical analyses of this Meta-analyses were performed with Review Manager 5.3 and Stata 15.0 software to conduct a Meta-analysis of the symptom improvement of BPH patients before and 3 months and 12 months after PUL. The main evaluation indicators included: International Prostate Symptom Score (IPSS), maximum urinary flow rate (Q max), post-void residual (PVR), and Quality of Life Scale (QOL), Sexual Health Inventory for Men (SHIM). The complication rate of PUL was systematically evaluated. Results:A total of 12 clinical studies were included, and 850 patients accepted the PUL. The results showed that IPSS decreased significantly at both 3 and 24 months after PUL surgery ( MD = -11.77, 95% CI -12.47—-11.07, P<0.05; MD = -9.71, 95% CI-10.76—-8.66, P<0.05), Q max (ml/s) increased to a certain degree ( MD = 3.87, 95% CI 3.37—4.37, P<0.05; MD = 3.68, 95% CI 2.97—4.40, P<0.05), QOL decreased significantly ( MD=-2.57, 95% CI -2.76—-2.38, P<0.05; MD = -2.14, 95% CI -2.38—-2.91, P<0.05), SHIM score was unaffected ( P>0.05), compared with preoperative baseline data. PUL could be performed under local anesthesia, the main perioperative complications reported in the included studies were dysuria (17%±6%), hematuria (14%±5%) and pelvic pain (8%±6%), all of which were transient. Conclusions:PUL in the treatment of BPH has significant short-term and long-term efficacy with low surgical risk and complication rate, and can preserve normal ejaculation function. It is a safe and effective minimally invasive surgery, which can be used for BPH patients with intolerance to general anesthesia surgery or normal sexual function demand.

Silicosis is a leading cause of occupational disease-related morbidity and mortality worldwide, but the molecular basis underlying its development remains unclear. An accumulating body of evidence supports gasdermin D (GSDMD)-mediated pyroptosis as a key component in the development of various pulmonary diseases. However, there is little experimental evidence connecting silicosis and GSDMD-driven pyroptosis. In this work, we investigated the role of GSDMD-mediated pyroptosis in silicosis. Single-cell RNA sequencing of healthy and silicosis human and murine lung tissues indicated that GSDMD-induced pyroptosis in macrophages was relevant to silicosis progression. Through microscopy we then observed morphological alterations of pyroptosis in macrophages treated with silica. Measurement of interleukin-1β release, lactic dehydrogenase activity, and real-time propidium iodide staining further revealed that silica induced pyroptosis of macrophages. Additionally, we verified that both canonical (caspase-1-mediated) and non-canonical (caspase-4/5/11-mediated) signaling pathways mediated silica-induced pyroptosis activation, in vivo and in vitro. Notably, Gsdmd knockout mice exhibited dramatically alleviated silicosis phenotypes, which highlighted the pivotal role of pyroptosis in this disease. Taken together, our results demonstrated that macrophages underwent GSDMD-dependent pyroptosis in silicosis and inhibition of this process could serve as a viable clinical strategy for mitigating silicosis.