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Current Protocols in Protein Science

Keyun Wang, Mingliang Ye
Protein methylation is receiving increasing attention for its important role in regulating diverse biological processes, including epigenetic regulation of gene transcription, RNA processing, DNA damage repair, and signal transduction. Proteome level analysis of protein methylation requires the enrichment of various forms of methylated peptides. Unfortunately, immunoaffinity purification can only enrich a subset of them due to the lack of pan-specific antibodies. Chromatography-based methods, however, can enrich methylated peptides in a global manner...
February 21, 2018: Current Protocols in Protein Science
Sarah L Irons, Adam C Chambers, Olga Lissina, Linda A King, Robert D Possee
Baculovirus expression systems are well established as an easy and reliable way to produce high quality recombinant proteins. Baculoviruses can also be used to transduce mammalian cells, termed 'BacMam', with considerable potential in biomedical applications. This chapter explains the process of making a recombinant baculovirus, encompassing production of a recombinant virus by homologous recombination in insect cells, followed by amplification and titration of the virus-all steps needed before commencing gene expression and protein production...
February 21, 2018: Current Protocols in Protein Science
Merideth A Cooper, Joseph E Taris, Changhua Shi, David W Wood
In this work, we describe a novel self-cleaving tag technology based on a highly modified split-intein cleaving element. In this system, the N-terminal segment of an engineered split intein is expressed in E. coli and covalently immobilized onto a capture resin, while the smaller C-terminal intein segment is fused to the N-terminus of the desired target protein. The tagged target can then be expressed in an appropriate expression system, without concern for premature intein cleaving. During the purification, strong association between the intein segments effectively captures the tagged target onto the capture resin while simultaneously generating a cleaving-competent intein complex...
February 21, 2018: Current Protocols in Protein Science
Debra T Hansen, Felicia M Craciunescu, Petra Fromme, Stephen A Johnston, Kathryn F Sykes
Membrane proteins are the molecular interface of the cell and its environs; however, studies of membrane proteins are highly technically challenging, mainly due to instability of the isolated protein. Towards the production of antibodies that recognize properly folded and stabilized forms of membrane protein antigen, we describe a DNA-based immunization method for mice that expresses the antigen in the membranes of dendritic cells, thus allowing direct presentation to the immune system. This genetic immunization approach employs a highly efficient method of biolistic delivery based on DNA-gold micronanoplexes, which are complexes of micron-sized gold particles that allow dermal penetration and nanometer-sized gold particles that provide a higher surface area for DNA binding than micron gold alone...
February 21, 2018: Current Protocols in Protein Science
Adam C Chambers, Mine Aksular, Leo P Graves, Sarah L Irons, Robert D Possee, Linda A King
This unit provides information on the replication cycle of insect baculovirus to provide an understanding of how this virus has been adapted for use as an expression vector for recombinant proteins in insect cells. We provide an overview of the virus structure and its unique bi-phasic replication cycle, which has been exploited in developing the virus as an expression vector. We also review the development of the baculovirus expression vector system (BEVS), from the mid-1980s to the present day in which the BEVS is now an established tool for the production of a range of recombinant proteins and multi-protein complexes including virus-like particles...
February 21, 2018: Current Protocols in Protein Science
Kyle J Roux, Dae In Kim, Brian Burke, Danielle G May
BioID is a unique method to screen for physiologically relevant protein interactions that occur in living cells. This technique harnesses a promiscuous biotin ligase to biotinylate proteins based on proximity. The ligase is fused to a protein of interest and expressed in cells, where it biotinylates proximal endogenous proteins. Because it is a rare protein modification in nature, biotinylation of these endogenous proteins by BioID fusion proteins enables their selective isolation and identification with standard biotin-affinity capture...
February 21, 2018: Current Protocols in Protein Science
Lynn A Beer, David W Speicher
The most commonly used types of gels for separating proteins are SDS gels, either in a 1-D format or as the second dimension of various 2-D separations, and the most common methods of visualizing proteins in these gels use protein binding dyes after fixing the proteins in the gel matrix. In recent years, there has been a continuing trend away from preparing staining solutions in the laboratory to using commercially available kits, which are convenient, save time, have defined shelf lives, and may provide greater reproducibility than stains formulated in research laboratories...
February 21, 2018: Current Protocols in Protein Science
Srinivas Jayanthi, Ravi Kumar Gundampati, Thallapuranam Krishnaswamy Suresh Kumar
Heparin, a member of the glycosaminoglycan family, is known to interact with more than 400 different types of proteins. For the past few decades, significant progress has been made to understand the molecular details involved in heparin-protein interactions. Based on the structural knowledge available from the FGF1-heparin interaction studies, we have designed a novel heparin-binding peptide (HBP) affinity tag that can be used for the simple, efficient, and cost-effective purification of recombinant proteins of interest...
November 1, 2017: Current Protocols in Protein Science
Lisette G G C Verhoef, Mark Wade
The number of intracellular protein-protein interactions (PPIs) far exceeds the total number of proteins encoded by the genome. Dynamic cellular PPI networks respond to external stimuli and endogenous metabolism in order to maintain homeostasis. Many PPIs are directly involved in disease pathogenesis and/or resistance to therapeutics; they therefore represent potential drug targets. A technology generally termed 'bimolecular complementation' relies on the physical splitting of a molecular reporter (such as a fluorescent or luminescent protein) and fusion of the resulting two fragments to a pair of interacting proteins...
November 1, 2017: Current Protocols in Protein Science
Eric J Steinmetz, Michele E Auldridge
The simplicity, speed, and low cost of bacterial culture make E. coli the system of choice for most initial trials of recombinant protein expression. However, many heterologous proteins are either poorly expressed in bacteria, or are produced as incorrectly folded, insoluble aggregates that lack the activity of the native protein. In many cases, fusion to a partner protein can allow for improved expression and/or solubility of a difficult target protein. Although several different fusion partners have gained favor, none are universally effective, and identifying the one that best improves soluble expression of a given target protein is an empirical process...
November 1, 2017: Current Protocols in Protein Science
Ineke Braakman, Lydia Lamriben, Guus van Zadelhoff, Daniel N Hebert
In this unit, protocols are provided for detection of disulfide bond formation in cultures of intact cells and in an in vitro translation system containing isolated microsomes or semi-permeabilized cells. First, the newly synthesized protein of interest is biosynthetically labeled with radioactive amino acids in a short pulse. The labeled protein then is chased with unlabeled amino acids. At different times during the chase, a sample is collected, membranes are lysed with detergent, and the protein is isolated by immunoprecipitation, as described...
November 1, 2017: Current Protocols in Protein Science
Irene Nigi, Louise Fairall, John W R Schwabe
Prokaryotic expression systems have been widely used to express proteins for structural studies. Such expression systems have the advantage of being economical, straightforward and fast. However, for many eukaryotic proteins and particularly protein complexes, bacterial expression systems do not produce significant yields of soluble protein. This may result from failure to efficiently transcribe/translate the required protein or may result from the formation of insoluble aggregates known as inclusion bodies...
November 1, 2017: Current Protocols in Protein Science
Jared Kim, Ho Leung Ng
This unit addresses several critical challenges associated with membrane protein crystallography by screening membrane proteins from Escherichia coli, Saccharomyces cerevisiae, and Sus scrofa cerebral tissue for biochemical properties favorable for crystallization. First, a tissue sample or cell pellet is obtained. The cells are isolated, washed, and then lysed either by sonication, bead beating, or manual homogenization. Membrane proteins are fractionated from the lysates by centrifugation and solubilized in a mild detergent suitable for crystallization, such as n-dodecyl-β-maltoside (DDM)...
November 1, 2017: Current Protocols in Protein Science
Catherine A Cotter, Patricia L Earl, Linda S Wyatt, Bernard Moss
The culturing of cell lines used with vaccinia virus, both as monolayer and in suspension, is described. The preparation of chick embryo fibroblasts (CEF) is presented for use in the production of the highly attenuated and host range-restricted modified vaccinia virus Ankara (MVA) strain of vaccinia virus. Protocols for the preparation, titration, and trypsinization of vaccinia virus stocks, as well as viral DNA preparation and virus purification methods are also included. © 2017 by John Wiley & Sons, Inc.
August 1, 2017: Current Protocols in Protein Science
Laurie Ceccato, Mélanie Mansat, Bernard Payrastre, Frédérique Gaits-Iacovoni, Julien Viaud
Phosphoinositides are key signaling and regulatory phospholipids that mediate important pathophysiological processes. This is achieved through the interaction of their phosphorylated inositol head group with a wide range of protein domains. Therefore, being able to determine the phosphoinositide specificity for effector protein is essential to the understanding of its cellular function. This unit describes a novel method named Protein-Lipid Interaction by Fluorescence, or PLIF. PLIF is a fast, reliable and high throughput assay that allows determination of the phosphoinositide specificity of proteins, simultaneously providing relative affinities...
August 1, 2017: Current Protocols in Protein Science
Avital Percher, Emmanuelle Thinon, Howard Hang
The covalent coupling of fatty acids to proteins provides an important mechanism of regulation in cells. In eukaryotes, cysteine fatty acylation (S-fatty acylation) has been shown to be critical for protein function in a variety of cellular pathways as well as microbial pathogenesis. While methods developed over the past decade have improved the detection and profiling of S-fatty acylation, these are hampered in their ability to characterize endogenous protein S-fatty acylation levels under physiological conditions...
August 1, 2017: Current Protocols in Protein Science
Monika B Dolinska, Paul T Wingfield, Yuri V Sergeev
The purification of an enzyme from insect larvae infected with a baculovirus vector is described. The enzyme tyrosinase is of biomedical importance and catalyzes the first rate-limiting steps in melanin production. Tyrosinase mutations can result in oculocutaneous albinism type 1 (OCA1), an inherited eye disease associated with decreased melanin pigment production and vision defects. To simplify expression and subsequent purification, the extracellular domain is expressed in insect cells, produced in Trichoplusia ni larvae, and purified using affinity and size-exclusion chromatography...
August 1, 2017: Current Protocols in Protein Science
Linda S Wyatt, Patricia L Earl, Bernard Moss
This unit describes how to infect cells with vaccinia virus and then transfect them with a plasmid-transfer vector or PCR fragment to generate a recombinant virus. Selection and screening methods used to isolate recombinant viruses and a method for the amplification of recombinant viruses are described. Finally, a method for live immunostaining that has been used primarily for detection of recombinant modified vaccinia virus Ankara (MVA) is presented. © 2017 by John Wiley & Sons, Inc.
August 1, 2017: Current Protocols in Protein Science
John M Antos, Jessica Ingram, Tao Fang, Novalia Pishesha, Matthias C Truttmann, Hidde L Ploegh
Strategies for site-specific protein modification are highly desirable for the construction of conjugates containing non-genetically-encoded functional groups. Ideally, these strategies should proceed under mild conditions, and be compatible with a wide range of protein targets and non-natural moieties. The transpeptidation reaction catalyzed by bacterial sortases is a prominent strategy for protein derivatization that possesses these features. Naturally occurring or engineered variants of sortase A from Staphylococcus aureus catalyze a ligation reaction between a five-amino-acid substrate motif (LPXTG) and oligoglycine nucleophiles...
August 1, 2017: Current Protocols in Protein Science
Jongchan Woo, Jason Hong, Savithramma P Dinesh-Kumar
Bioluminescence resonance energy transfer (BRET) is a technique that analyzes protein-protein interactions (PPIs). The unique feature of BRET delineates that the resonance energy is generated by the resonance energy donor, Renilla luciferase by the oxidative decarboxylation of coelenterazine substrate. BRET is superior to FRET where issues such as autofluorescence, photobleaching, and light scattering can occur. Recently, BRET has been applied to design synthetic biosensors for monitoring autophagy in vivo and in vitro...
April 3, 2017: Current Protocols in Protein Science
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