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affinity Chromatography purification of Recombinant Human Interleukin-6 from Its Fusion Protein with GST

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Transactions of Tianjin University 2002年第2期8卷 106-109页

作者：吴蕾甘一如林峰黄鹤天津大学化工学院天津300072

Recombinant *** JM109, containing pHZ1818 plasmid which included the fused gene encoding human interleukin 6(IL 6), expressed a fusion protein with glutathion S transferase(GST). The fusion protein existed both in the supernatant and inside the bacterial cell,but the insoluble protein had no biological activity and could not be refolded. The rotative speed of the shaker and the temperature of induction were optimized to maximize the expression of the soluble fusion protein. From the supernatant of the cell sonicates Glutathion Sephrose 4B affinity column chromatography was employed to isolate the fusion protein which could be purified to >80 0 0 in a single step. The yield of soluble GST IL 6 was about 10 mg per liter culture. The GST was site specifically cloven by 6 hours of treatment with thrombin and from the thrombin digest mixture IL 6 was purified by Q high performance ion exchange chromatography. From 1 liter of *** culture 2 mg refined IL 6 was obtained. The purified IL 6 had a purity of more than 95 0 0 and a biological activity of 1.02×10 8 IU/mg.

关键词： affinity purification human interleukin 6 fusion protein GST

Recent progress in mass spectrometry proteomics for biomedical research

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Science China(Life Sciences) 2017年第10期60卷 1093-1113页

作者： Xu Li Wenqi Wang Junjie Chen Department of Experimental Radiation Oncology The University of Texas MD Anderson Cancer Center Houston TX 77030 USA Institute of Biology Westlake Institute for Advanced Study Westlake University Los Angeles CA 91604 USA Department of Developmental and Cell Biology University of California Irvine CA 92697 USA

Proteins are the key players in many cellular processes. Their composition, trafficking, and interactions underlie the dynamic processes of life. Furthermore, diseases are frequently accompanied by malfunction of proteins at multiple levels. Understanding how biological processes are regulated at the protein level is critically important to understanding the molecular basis for diseases and often shed light on disease prevention, diagnosis, and treatment. With rapid advances in mass spectrometry(MS)instruments and experimental methodologies, MS-based proteomics has become a reliable and essential tool for elucidating biological processes at the protein level. Over the past decade, we have witnessed great expansion of knowledge of human diseases with the application of MS-based proteomic technologies, which has led to many exciting discoveries. Herein we review the recent progress in MS-based proteomics in biomedical research, including that in establishing disease-related proteomes and interactomes. We also discuss how this progress will benefit biomedical research and clinical diagnosis and treatment of disease.

关键词： affinity purification LC-MS/MS mass spectrometry shotgun proteomics

Preparation of Mycobacterium smegmatis porin A(MspA)nanopores for single molecule sensing of nucleic acids

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Biophysics Reports 2021年第5期7卷 355-364页

作者： Yuqin Wang Pingping Fan Shanyu Zhang Shuanghong Yan Shuo Huang State Key Laboratory of Analytical Chemistry for Life Sciences School of Chemistry and Chemical EngineeringNanjing University Chemistry and Biomedicine Innovation Center (ChemBIC) Nanjing University

Mycobacterium smegmatis porin A(MspA), possessing a short(～0.6 nm) and narrow(～1.2 nm) constriction that enables its high spatial resolution of sensing, has emerged as an optimum choice for nanopore sequencing and nano-reactive sensing. However, prepared Msp A nanopores cannot be obtained from any commercial vendors. We here provide a highly simplified protocol for Msp A preparation. The protocol yields ～10 mg fully oligomerized protein per liter of culture and is straightforward to *** the prepared Msp A nanopores, discrimination of immobilized ss DNA oligonucleotides can be performed, which serves as a demo application to demonstrate core procedures of single molecule sensing using MspA. This method is also in principle compatible with all other Msp A mutants, which might merit the need to develop a variety of MspA based nano-reactive sensors.

关键词： MspA nanopore Prokaryotic expression affinity purification Single molecule sensing

Exploring the Nicotinic Acetylcholine Receptor-associated Proteome with iTRAQ and Transgenic Mice

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Genomics, Proteomics & Bioinformatics 2013年第4期11卷 207-218页

作者： Tristan D. McClure-Begley Kathy L. Stone Michael J. Marks Sharon R. Grady Christopher M. Colangelo Jon M. Lindstrom Marina R. Picciotto Department of Psychiatry Yale University School of MedicineNew Haven CT 06508 USA Institute for Behavioral Genetics University of ColoradoBoulder CO 80303 USA W.M. Keck Biotechnology Resource Laboratory Yale University School MedicineNew Haven CT 06509 USA Department of Psychology and Neuroscience University of ColoradoBoulder CO 80309 USA Department of Molecular Biophysics & Biochemistry Yale UniversityNew Haven CT 06520 USA Department of Neuroscience Medical School of the University of PennsylvaniaPhiladelphia PA 19104 USA

Neuronal nicotinic acetylcholine receptors （nAChRs） containing Gt4 and 132 subunits are the principal receptors in the mammalian central nervous system that bind nicotine with high affin- ity. These nAChRs are involved in nicotine dependence, mood disorders, neurodegeneration and neuroprotection. However, our understanding of the interactions between a4β2-containing （a4β2） nAChRs and other proteins remains limited. In this study, we identified proteins that inter- act with ct4β2 nAChRs in a gene-dose dependent pattern by immunopurifying β2 nAChRs from mice that differ in ct4 and β2 subunit expression and performing proteomic analysis using isobaric tags for relative and absolute quantitation （iTRAQ）. Reduced expression of either the a4 or the β2 subunit results in a correlated decline in the expression of a number of putative interacting proteins. We identified 208 proteins co-imrnunoprecipitated with these nAChRs. Furthermore, stratified lin- ear regression analysis indicated that levels of 17 proteins was correlated significantly with expres- sion of at4β2 nAChRs, including proteins involved in cytoskeletal rearrangement and calcium signaling. These findings represent the first application of quantitative proteomics to produce a β2 nAChR interactome and describe a novel technique used to discover potential targets for pharma- cological manipulation of a4β2 nAChRs and their downstream signaling mechanisms.

关键词： Nicotinic receptor affinity purification Quantitative proteomics Transgenic mouse