Silica-supported Macroporous Chitosan Bead for Affinity Purification of Trypsin Inhibitor

Macroporous cross-linking chitosan layer coated on silica gel （CTS-SiO2） was prepared by phase inversion and polyethylene glycol （PEG）molecular imprinting methods. Formation of macroporous surtace was investigated by scanning electron microscopy （SEM） and BET analysis.The prepared bead was activated by reacting with 1,2-ethylene digiycidyl ether for introducing epoxy groups, and trypsin could be efficiently immobilized on the bead as a biospecific ligand.The bead bearing trypsin was employed to purify trypsin inhibitor （TIs） from egg white as affinity adsorbent.

Design of Ligands for Affinity Purification of G-CSF Based on Peptide Ligands Derived from a Peptide Library

Combinatorial peptide libraries have become powerful tools to screen functional ligands by the principle of affinity selection. We screened in a phage peptide library to investigate potential peptide affinity ligands for the purification of human granulocyte colonystimulation factor(hGCSF). Peptide ligands will be promising to replace monoclonal antibodies as they have advantages of high stability, efficiency, selectivity and low price.

Affinity Chromatography Purification of Recombinant Human Interleukin-6 from Its Fusion Protein with GST

Recombinant E.coli 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 E.coli 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.

SYNTHESIS OF INTERFERON-α_A MONOCLONAL ANTIBODY PACKING MATERIAL IN HIGH-PERFORMANCE AFFINITY CHROMATOGRAPHY AND PURIFICATION OF RECOMBINANT HUMAN INTERFERON-α_A

A new way for the synthesis of human interferon—α_A monoclonal antibody (IFN-α_A-McAb) bound to silica gel packing material in high-performance affinity chromatography (HPAFC) has been developed. The high coupling efficiency and specific activity of IFN—α_A-McAb can be obtained by activated diol-silica gel with activating agent. After purification using this packing material in HPAFC, the specific activity of recombinant human interferon-α_A (rIFN-α_A) rose up to 1.03×10~7IU/mg protein and the purification efficiency is appoximately 100 times.

Research Progress of Using Phage Display Technology to Screen Virus Affinity Peptides

Development and application of phage display technology and research progress of virus affinity peptide were summarized in the paper,and a preliminary outlook for future development was put forward. The paper laid a foundation for development of polypeptide drugs and polypeptide vaccine.

RUVBL2, a novel AS160-binding protein, regulates insulinstimulated GLUT4 translocation

In fat and muscle cells, insulin-stimulated glucose uptake is mainly mediated by glucose transporter 4 （GLUT4）, which translocates from intracellular compartments to the cell surface in response to insulin stimulation. AS160 is one of the substrates of Akt and plays important roles in insulin-regulated GLUT4 translocation. In this study, RuvB- like protein 2 （RUVBL2） is identified as a new AS160-binding protein using mammalian tandem affinity purification （TAP） combined with mass spectrometry. In 3T3-L1 adipocytes, RUVBL2 is highly expressed and is mainly distrib- uted in the cytosol. Depletion of RUVBL2 in adipocytes inhibits insulin-stimulated GLUT4 translocation and glucose uptake through reducing insulin-stimulated ASI60 phosphorylation. However, introduction of human RUVBL2 can reverse this inhibitory effect. These data suggest that RUVBL2 plays an important role in insulin-stimulated GLUT4 translocation through its interaction with AS160.

Expression of Overlapping PCR-generated Shiga Toxin B Gene Fragment in E. Coli and Its Ascitic Polyclonal Antibody

The mature Shiga toxin B （StxB） gene was optimized and generated by overlapping PCR. Recombinant expression vector pQE40-DHFR/StxB was constructed when the gene was cloned into pQE fusion expression vector. Induced by Isopropyl β-D-thiogalactoside （IPTG）, the DHFR/StxB fusion protein was highly expressed to the level of 41.36% in E. coli MI5 cells. The 35 kDa fusion protein with a 6 His-tag was one-step purified from inclusion bodies using Ni-NTA affinity chromatography column under denaturing conditions, and was refolded by dialyzing with a decreasing urea gradient. Purified DHFR/StxB fusion protein was used to immunize Kunming mice for generating the ascitic polyclonal antibody against recombinant StxB protein by injecting sarcoma 180 cells and the titer ofascitic polyclonal antibody is up to 1： 1× 10^6 detected by the indirect enzyme linked immunosorbent assy （ELISA）. Western immunoblotting analysis revealed that the ascitic polyclonal antibody against StxB had a specific affinity for a 70 kDa shiga toxin protein of Shigella dysenteriae type 1. It is a new simple and quick method to produce a large amount of ascitic polyclonal antibody. The antibody is used to develop immunological method for detecting shiga toxin.

Newfound Coding Potential of Transcripts Unveils Missing Members of Human Protein Communities

Recent proteogenomic approaches have led to the discovery that regions of the transcriptome previously annotated as non-coding regions[i.e.,untranslated regions(UTRs),open reading frames overlapping annotated coding sequences in a different reading frame,and non-coding RNAs]frequently encode proteins,termed alternative proteins(altProts).This suggests that previously identified protein–protein interaction(PPI)networks are partially incomplete because altProts are not present in conventional protein databases.Here,we used the proteogenomic resource OpenProt and a combined spectrum-and peptide-centric analysis for the re-analysis of a highthroughput human network proteomics dataset,thereby revealing the presence of 261 altProts in the network.We found 19 genes encoding both an annotated(reference)and an alternative protein interacting with each other.Of the 117 altProts encoded by pseudogenes,38 are direct interactors of reference proteins encoded by their respective parental genes.Finally,we experimentally validate several interactions involving altProts.These data improve the blueprints of the human PPI network and suggest functional roles for hundreds of altProts.

Recent progress in mass spectrometry proteomics for biomedical research

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.

Reproductive tissue-specific translatome of a rice thermo-sensitive genic male sterile line

Translational regulation,especially tissue-or cell type-specific gene regulation,plays essential roles in plant growth and development.Thermo-sensitive genic male sterile(TGMS)lines have been widely used for hybrid breeding in rice(Oryza sativa).However,little is known about translational regulation during reproductive stage in TGMS rice.Here,we use translating ribosome affinity purification(TRAP)combined with RNA sequencing to investigate the reproductive tissue-specific translatome of TGMS rice expressing FLAG-tagged ribosomal protein L18(RPL18)from the germline-specific promoter MEIOSIS ARRESTED AT LEPTOTENE1(MEL1).Differentially expressed genes at the transcriptional and translational levels are enriched in pollen and anther-related formation and development processes.These contain a number of genes reported to be involved in tapetum programmed cell death(PCD)and lipid metabolism during pollen development and anther dehiscence in rice,including several encoding transcription factors and key enzymes,as well as several long non-coding RNAs(lnc RNAs)that potentially affect tapetum and pollenrelated genes in male sterility.This study represents the comprehensive reproductive tissue-specific characterization of the translatome in TGMS rice.These results contribute to our understanding of the molecular basis of sterility in TGMS rice and will facilitate further genetic manipulation of TGMS rice in two-line breeding systems.

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

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.