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Current Protocols in Protein Science

Guoping Ren, Na Ke, Mehmet Berkmen
Escherichia coli continues to be a popular expression host for the production of proteins, yet successful recombinant expression of active proteins to high yields remains a trial and error process. This is mainly due to decoupling of the folding factors of a protein from its native host, when expressed recombinantly in E. coli. Failure to fold could be due to many reasons but is often due to lack of post-translational modifications that are absent in E. coli. One such post-translational modification is the formation of disulfide bonds, a common feature of secreted proteins...
2016: Current Protocols in Protein Science
Norio Yamamoto, Yoko Yamashita, Yasukiyo Yoshioka, Shin Nishiumi, Hitoshi Ashida
Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells...
2016: Current Protocols in Protein Science
Daniel Ivanusic, Joachim Denner, Norbert Bannert
This unit provides a guide and detailed protocol for studying membrane protein-protein interactions (PPI) using the acceptor-sensitized Förster resonance electron transfer (FRET) method in combination with the proximity ligation assay (PLA). The protocol in this unit is focused on the preparation of FRET-PLA samples and the detection of correlative FRET/PLA signals as well as on the analysis of FRET-PLA data and interpretation of correlative results when using cyan fluorescent protein (CFP) as a FRET donor and yellow fluorescent protein (YFP) as a FRET acceptor...
2016: Current Protocols in Protein Science
Björn Koos, Ola Söderberg
Proximity-dependent hybridization chain reaction (proxHCR) is a novel technique for detection of protein interaction, post-translational modifications (PTMs), or protein expression. The method is based upon antibodies targeting the proteins of interest that are covalently conjugated to DNA oligonucleotides, which enables the induction of a hybridization chain reaction (HCR) to generate a fluorescent signal visible under a microscope. In contrast to the in situ proximity ligation assay (in situ PLA), which is another method that utilizes antibody-DNA conjugates to detect protein interactions, proxHCR does not require enzymatic steps...
2016: Current Protocols in Protein Science
Laurent Kreplak
The atomic force microscope (AFM) has the unique capability of imaging biological samples with molecular resolution in buffer solution over a wide range of time scales from milliseconds to hours. In addition to providing topographical images of surfaces with nanometer- to angstrom-scale resolution, forces between single molecules and mechanical properties of biological samples can be investigated from the nano-scale to the micro-scale. Importantly, the measurements are made in buffer solutions, allowing biological samples to "stay alive" within a physiological-like environment while temporal changes in structure are measured-e...
2016: Current Protocols in Protein Science
Colin T Mant, Robert S Hodges
Reversed-phase high-performance liquid chromatography (RP-HPLC) is of fundamental importance to the isolation and separation of peptides, proteins, and other biomolecules. Hence, there is a continuing high demand for the development of RP-HPLC stationary-phase materials with enhanced separation efficiency. HALO packing materials began the revolution in "core-shell" technology with the advantages of faster separations, higher resolution and peak capacity, high temperature stability, and rugged reliable performance compared to traditional HPLC and UHPLC...
2016: Current Protocols in Protein Science
(no author information available yet)
The basic theory of protein precipitation by addition of ammonium sulfate is presented, and the most common applications are listed. Tables are provided for calculating the appropriate amount of ammonium sulfate to add to a particular protein solution.
2016: Current Protocols in Protein Science
(no author information available yet)
The reagents and methods for purification and use of the most commonly used denaturants, guanidine hydrochloride (guanidine-HCl) and urea, are described. Other protein denaturants and reagents used to fold proteins are briefly mentioned. Sulfhydryl reagents (reducing agents) and "oxido-shuffling" (or oxidative regeneration) systems are also described.
2016: Current Protocols in Protein Science
Michael A Massiah, Katharine M Wright, Haijuan Du
This unit describes a straightforward and efficient method of using sarkosyl to solubilize and recover difficult recombinant proteins, such as GST- and His6 -tagged fusion proteins, that are overexpressed in E. coli. This protocol is especially useful for rescuing recombinant proteins overexpressed in M9 minimal medium. Sarkosyl added to lysis buffers helps with both protein solubility and cell lysis. Higher percentage sarkosyl (up to 10%) can extract >95% of soluble protein from inclusion bodies. In the case of sarkosyl-solubilized GST-fusion proteins, batch-mode affinity purification requires addition of a specific ratio of Triton X-100 and CHAPS, while sarkosyl-solubilized His6 -tagged fusion proteins can be directly purified on Ni(2+) resin columns...
2016: Current Protocols in Protein Science
Marcella Orwick-Rydmark, Thomas Arnold, Dirk Linke
Extraction of membrane proteins from biological membranes is usually accomplished with the help of detergents. This unit describes the use of detergents to solubilize and purify membrane proteins. The chemical and physical properties of the different classes of detergents typically used with biological samples are discussed. A separate section addresses the compatibility of detergents with applications downstream of the membrane protein purification process, such as optical spectroscopy, mass spectrometry, protein crystallography, biomolecular NMR, or electron microscopy...
2016: Current Protocols in Protein Science
Li Lei, Martin Egli
Fish and human cytochrome P450 (P450) 17A1 catalyze both steroid 17α-hydroxylation and 17α,20-lyase reactions. Fish P450 17A2 catalyzes only 17α-hydroxylation. Both enzymes are microsomal-type P450s, integral membrane proteins that bind to the membrane through their N-terminal hydrophobic segment, the signal anchor sequence. The presence of this N-terminal region renders expression of full-length proteins challenging or impossible. For some proteins, variable truncation of the signal anchor sequence precludes expression or results in poor expression levels...
2016: Current Protocols in Protein Science
Daumantas Matulis
Selective precipitation of proteins can be used as a bulk method to recover the majority of proteins from a crude lysate, as a selective method to fractionate a subset of proteins from a protein solution, or as a very specific method to recover a single protein of interest from a purification step. This unit describes a number of methods suitable for selective precipitation. In each of the protocols that are outlined, the physical or chemical basis of the precipitation process, the parameters that can be varied for optimization, and the basic steps for developing an optimized precipitation are described...
2016: Current Protocols in Protein Science
Constance J Jeffery
Bacterial integral membrane proteins play many important roles, including sensing changes in the environment, transporting molecules into and out of the cell, and in the case of commensal or pathogenic bacteria, interacting with the host organism. Working with membrane proteins in the lab can be more challenging than working with soluble proteins because of difficulties in their recombinant expression and purification. This protocol describes a standard method to express, solubilize, and purify bacterial integral membrane proteins...
2016: Current Protocols in Protein Science
Michal Zolkiewski, Liudmila S Chesnokova, Stephan N Witt
Protein aggregation is a common problem in protein biochemistry and is linked to many cellular pathologies and human diseases. The molecular chaperone ClpB can resolubilize and reactivate aggregated proteins. This unit describes the procedure for following reactivation of an aggregated enzyme glucose-6-phosphate dehydrogenase mediated by ClpB from Escherichia coli in cooperation with another molecular chaperone, DnaK. The procedures for purification of these chaperones are also described.
2016: Current Protocols in Protein Science
Sven Fridrich, Konstantin Karmilin, Walter Stöcker
Substrate cleavage by metalloproteinases involves nucleophilic attack on the scissile peptide bond by a water molecule that is polarized by a catalytic metal, usually a zinc ion, and a general base, usually the carboxyl group of a glutamic acid side chain. The zinc ion is most often complexed by imidazole nitrogens of histidine side chains. This arrangement suggests that the physiological pH optimum of most metalloproteinases is in the neutral range. In addition to their catalytic metal ion, many metalloproteinases contain additional transition metal or alkaline earth ions, which are structurally important or modulate the catalytic activity...
2016: Current Protocols in Protein Science
Kevin D Sarge
Sumoylation, wherein small ubiquitin-like modifier (SUMO) proteins are covalently attached to specific lysine residues of target proteins, plays an important role in regulating many diverse cellular processes via its control of the functional properties of the modified proteins. Identification of new sumoylated proteins is expected to expand understanding of the role this modification has in cell function. This unit describes two different assays for determining whether a particular protein is sumoylated: the first method employs immunoprecipitation of the protein followed by SUMO immunoblot...
2016: Current Protocols in Protein Science
Kathrine B Sylvestersen, Michael L Nielsen
The attachment of one or more methylation groups to the side chain of arginine residues is a regulatory mechanism for cellular proteins. Recent advances in mass spectrometry-based characterization allow comprehensive identification of arginine methylation sites by peptide-level enrichment strategies. Described in this unit is a 4-day protocol for enrichment of arginine-methylated peptides and subsequent identification of thousands of distinct sites by mass spectrometry. Specifically, the protocol explains step-by-step sample preparation, enrichment using commercially available antibodies, prefractionation using strong cation exchange, and identification using liquid chromatography coupled to tandem mass spectrometry...
November 2, 2015: Current Protocols in Protein Science
James Chun Yip Chan, Lei Zhou, Eric Chun Yong Chan
The isotope-coded affinity tag (ICAT) technique has been applied to measure pairwise changes in protein expression through differential stable isotopic labeling of proteins or peptides followed by identification and quantification using a mass spectrometer. Changes in protein expression are observed when the identical peptide from each of two biological conditions is identified and a difference is detected in the measurements comparing the peptide labeled with the heavy isotope to the one with a normal isotopic distribution...
November 2, 2015: Current Protocols in Protein Science
David M Belnap
Macromolecular electron microscopy typically depicts the structures of macromolecular complexes ranging from ∼200 kDa to hundreds of MDa. The amount of specimen required, a few micrograms, is typically 100 to 1000 times less than needed for X-ray crystallography or nuclear magnetic resonance spectroscopy. Micrographs of frozen-hydrated (cryogenic) specimens portray native structures, but the original images are noisy. Computational averaging reduces noise, and three-dimensional reconstructions are calculated by combining different views of free-standing particles ("single-particle analysis")...
November 2, 2015: Current Protocols in Protein Science
Aaron Goldman, Jeanine A Ursitti, Jacek Mozdzanowski, David W Speicher
Transferring proteins from polyacrylamide gels onto retentive membranes is now primarily used for immunoblotting. A second application that was quite common up to about a decade ago was electroblotting of proteins for N-terminal and internal sequencing using Edman chemistry. This unit contains procedures for electroblotting proteins from polyacrylamide gels onto a variety of membranes, including polyvinylidene difluoride (PVDF) and nitrocellulose. In addition to the commonly used tank or wet transfer system, protocols are provided for electroblotting using semidry and dry systems...
November 2, 2015: Current Protocols in Protein Science
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