bluuebunny/crossref_metadata_2025_split huggingface dataset:
Use of the Gateway System for Protein Expression in Multiple Hosts
Authors: Hartley James L. 
Published: June 2004

AbstractThe Gateway cloning method allows a gene to be cloned and subsequently transferred into any vector by in vitro site‐specific recombination. It does not necessarily follow, however, that a gene can be cloned once and expressed in all the available host‐vector combinations with uniformly satisfactory results. This is because different organisms have different mechanisms of translating mRNA into protein, and also because choices always have to be made when designing an expression construct, for example, the presence or absence of a stop codon. This unit reviews Gateway cloning, summarizes aspects of protein expression that limit the universality of the use of one clone in many vectors and hosts, and discusses how conflicts between the structure of a Gateway clone of a gene and the rules of protein expression can be minimized or resolved.The Gateway cloning method allows a gene to be cloned and subsequently transferred into any vector by in vitro site‐specific.
Preparation of Soluble Proteins fromEscherichia coli
Authors: Wingfield Paul T. 
Published: June 2004

AbstractPurification of human IL‐1β is used in this unit as an example of the preparation of soluble proteins fromE. coli. Bacteria containing IL‐1β are lysed, and IL‐1 β in the resulting supernatant is purified by anion‐exchange chromatography, salt precipitation and cation‐exchange chromatography, and then concentrated. Finally, the IL‐1 β protein is applied to a gel‐filtration column to separate it from remaining higher‐ and lower‐molecular‐weight contaminants, the purified protein is stored frozen or is lyophilized. The purification protocol described is typical for a protein that is expressed in fairly high abundance (i.e., >5% total protein) and accumulates in a soluble state.
Preparation and Extraction of Insoluble (Inclusion‐Body) Proteins from Escherichia coli
Authors: Palmer Ira , Wingfield Paul T. 
Published: June 2004

AbstractHigh‐level expression of many recombinant proteins in Escherichia coli leads to the formation of highly aggregated protein commonly referred to as inclusion bodies. Inclusion bodies are normally formed in the cytoplasm; alternatively, if a secretion vector is used, they can form in the periplasmic space. Inclusion bodies can be recovered from cell lysates and this unit describes preparation of washed pellets and solubilization of the protein using guanidine⋅HCl. The extracted protein, which is unfolded, is either directly folded as described in UNIT here or further purified by gel filtration in the presence of guanidine⋅HCl as idescribed here. A support protocol describes the removal of guanidine⋅HCl from column fractions so they can be monitored by SDS‐PAGE.
Expression and Purification of GST Fusion Proteins
Authors: Harper Sandra , Speicher David W. 
Published: June 2004

AbstractAn increasingly common strategy for expressing proteins and large peptides in prokaryotic systems is to express the protein of interest connected to a “tag” that provides the basis for rapid high‐affinity purification. This unit describes the expression and purification of fusion proteins containing the 26‐kDa glutathione‐S‐transferase protein as well as methods for cleaving the affinity tag and repurifying the target protein. Advantages of this popular fusion protein system include high protein yields, high‐affinity one‐step protein purification of the fusion protein, existence of several alternative protease cleavage sites for removing the affinity tag when required, and ease of removal of the cleaved affinity tag.
Expression and Purification of Thioredoxin Fusion Proteins
Authors: McCoy John , La Ville Edward 
Published: June 2004

AbstractThis unit describes a gene fusion expression system that uses thioredoxin, the product of the Escherichia coli trxA gene, as the fusion partner. The inherent thermal stability of thioredoxin and its susceptibility to quantitative release from the E. coli cytoplasm by osmotic shock can also be exploited as useful tools for thioredoxin fusion protein purification. In this protocol, a fusion of trxA to any desired gene is constructed and the resulting fusion protein is expressed in an appropriate host strain. Additional protocols describe E. coli cell lysis and fractionation, osmotic release of thioredoxin fusion proteins from the E. coli cytoplasm, and heat treatment to purify some thioredoxin fusion proteins.
Overview of the Characterization of Recombinant Proteins
Authors: Denslow Nancy D. , Wingfield Paul T. , Rose Keith 
Published: June 2004

AbstractThis overview provides guidelines for the characterization of recombinantly expressed proteins (e.g., verifying primary structure and appropriate post‐translational modifications), along with methodologies for characterizing the proteins according to size, X‐ray structure, absorbance, biological activity, and subunit structure. A flow chart presents a suggested path for fully characterizing recombinant protein and involves equipment for HPLC, mass spectrometry, circular dichroism, NMR and fluorescence spectroscopy. Also covered are sources and consequences of contamination in protein solutions.
Determining the Identity and Purity of Recombinant Proteins by UV Absorption Spectroscopy
Authors: Mach Henryk , Middaugh C. Russell , Denslow Nancy 
Published: June 2004

AbstractBecause each protein (gene product) has a unique amino acid sequence, the particular aromatic amino acid content of each protein results in a unique spectrum in the near‐UV (250 to 350 nm) region. The highly specific microenvironment experienced by each aromatic residue in the three‐dimensional protein matrix results in fine shifts in a protein's spectrum. This unit provides protocols for the detection and analysis of UV spectra of recombinant proteins and their peptide fragments. The unique UV spectral properties of proteins can in turn be used to assess their purity. This application is inherent in the use of a diode array detector to monitor the effluent from a high‐performance liquid chromatography (HPLC) column. A protocol using this technique to assess the purity of recombinant proteins is presented.
Determining the Identity and Structure of Recombinant Proteins
Authors: Denslow Nancy D. , Rose Keith , Righetti Pier Giorgio 
Published: June 2004

AbstractIn this unit peptide mapping protocols with separation of the constituent peptides by high‐performance liquid chromatography (HPLC) analysis and by high‐resolution SDS‐PAGE are presented. Peptide mapping is ideally suited for comparative purposes‐‐for example, combined analysis of the recombinant protein and its natural counterpart (or some other well‐characterized standard). This unit also outlines the general strategy used to determine the linkage pattern of a monomeric recombinant protein containing two intramolecular disulfide bonds. The approach is an extension of peptide mapping, where the aim is to isolate and characterize peptides containing only a single disulfide bond. A two‐dimensional electrophoretic method is also described in which the protein isoelectric point is displayed as a function of pH to yield an electrophoretic titration curve. This method is especially useful for checking for deamidation (e.g., of Asn to Asp) in which additional negative charge is introduced into the modified protein.
Transverse Urea‐Gradient Gel Electrophoresis
Authors: Goldenberg David P. 
Published: June 2004

AbstractMonitoring the cooperative unfolding transition induced when a protein is exposed to elevated temperature or a chemical denaturant is an important strategy for characterizing the conformational properties of a globular protein. This transition may be analyzed quantitatively by a variety of spectroscopic techniques, but a simpler alternative is described in this unit: urea‐gradient gel electrophoresis. The pattern produced in the resulting gel can be used to estimate both the free energy change for unfolding and the rate of the unfolding transition. In addition, the technique can help identify either covalent or conformational heterogeneity in a protein sample. Because urea‐gradient gel patterns are sensitive to several parameters, including hydrodynamic volume, net charge, and conformational stability, the technique can be particularly useful for comparing two forms of a protein, e.g., a natural form and the product of recombinant bacteria.
Measuring Protein Thermostability by Differential Scanning Calorimetry
Authors: Makhatadze George I. 
Published: June 2004

AbstractDifferential scanning calorimetry (DSC) the method of choice to study the conformational stability of biological macromolecules and proteins in particular. This unit presents step‐by‐step protocols for DSC, including sample preparation and interpretation of the results in the simplest cases as well as calibration of the apparatus and maintenance of DSC cells. Various general experimental considerations, including possible pitfalls and errors, are also discussed.
One‐Dimensional Isoelectric Focusing of Proteins in Slab Gels
Authors: Ploegh Hidde L. 
Published: June 2004

AbstractThis unit describes isoelectric focusing (IEF) of polypeptides in urea‐containing polyacrylamide slab gels as a result of a pH gradient created by ampholytes (small charged organic molecules) in the gel in response to an electric field. Separation on the basis of isoelectric point produces bands of polypeptides that can be electrotransferred to suitable membranes for further analysis, for example, by staining or immunodetection. If samples have been radiolabeled, they can be visualized by fluorography. This unit contains support protocols detailing special conditions for application of denaturing IEF slab gels for electrotransfer and fluorography in which a scintillator is incorporated into the gel after completion of the run to facilitate detection of radioactively labeled polypeptides.
Electroblotting from Polyacrylamide Gels
Authors: Ursitti Jeanine A. , Mozdzanowski Jacek , Speicher David W. 
Published: June 2004

AbstractThis unit contains procedures for electrophoretically transferring proteins onto a variety of membranes including polyvinylidene difluoride (PVDF) and nitrocellulose, and derivatized membranes. The choice of membrane type for electrotransfer is dependent on the ultimate application for the blot membrane. An alternate protocol is provided for electroblotting in semidry systems. This unit also describes procedures for eluting proteins from membranes using detergents or acidic extraction with organic solvents.
Detection of Proteins on Blot Membranes
Authors: Harper Sandra , Speicher David W. 
Published: June 2004

AbstractStaining of blot transfer membranes permits visualization of proteins and allows the extent of transfer to be monitored. In the protocols described in this unit, proteins are stained after electroblotting from one‐dimensional or two‐dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluoride (PVDF), nitrocellulose, or nylon membranes. Protocols are provided for the use of six general protein stains:
amido black, Coomassie blue, Ponceau S, colloidal gold, colloidal silver, and India ink. In addition, the fluorescent stains fluorescamine and IAEDANS, which covalently react with bound proteins, are described. Approximate detection limits for each nonfluorescent stain are indicated along with membrane compatibilities.
Autoradiography
Authors: Bundy Daniel C. 
Published: June 2004

AbstractThis overview provides a thorough discussion of the theory and application of autoradiography for determining the spatial distribution of radioisotope‐labeled substances within a membrane or specimen material. The various types of film are discussed along with instructions on their correct handling. Quantitative autoradiography is described as a means of calibrating experimental results. Importantly, specific guidelines are given for pre‐flashing film, along with exposure and processing procedures.
Digital Electrophoresis Analysis
Authors: Medberry Scott , Gallagher Sean 
Published: June 2004

AbstractGel electrophoresis has become a ubiquitous method in molecular biology for separating biomolecules. This prominence is the result of several factors, including the robustness, speed, and potentially high throughput of the technique. The results of this method are traditionally documented using silver halide‐based photography followed by manual interpretation. While this remains an excellent method for qualitative documentation of single‐gel results, digital capture offers a number of significant advantages when documentation requires quantitation and sophisticated analysis. Digital images of gel electropherograms can be obtained rapidly using an image‐capture device, and the images can be easily manipulated using image analysis software. This overview presents reasons for digital documentation and analysis, defines some important key terms for imaging, explains the capture process and reviews the devices used for image capture, and provides an introduction to the software and methods used for one‐ and two‐dimensional digital image analysis.
Enzymatic Digestion of Proteins in Solution
Authors: Riviere Lise R. , Tempst Paul 
Published: June 2004

AbstractAnalysis of protein covalent structure is less complex and more accurate when performed on peptides derived from the larger protein. In contrast to acid‐promoted total hydrolysis, peptides are typically generated by selective proteolysis, i.e., by specifically cleaving peptide bonds with endoproteases that have varying degrees of specificity. This unit presents a protocol that can be used to generate peptide fragments from intact, undenatured proteins. Fragments can be analyzed directly by mass spectrometry (MS) or, more often, are first separated by reversed‐phase HPLC (RP‐HPLC) and then analyzed by MS or automated sequencing. Most proteins are resistant to enzymatic proteolysis under nondenaturing conditions or are not soluble in aqueous solution. Digestion procedures performed in the presence of chaotropic agents and SDS are described, and support protocols provide instructions for preparing enzyme stocks and reducing and alkylating peptides prior to sequencing or HPLC analysis.
Chemical Cleavage of Proteins on Membranes
Authors: Crimmins Dan L. , Mische Sheenah M. , Denslow Nancy D. 
Published: June 2004

AbstractDescribed in this unit are five basic protocols that are widely used for specific and efficient chemical cleavage of proteins bound to membranes. Cyanogen bromide (CNBr) cleaves at methionine (Met) residues; BNPS‐skatole cleaves at tryptophan (Trp) residues; formic acid cleaves at aspartic acid‐proline (Asp‐Pro) peptide bonds; hydroxylamine cleaves at asparagine‐glycine (Asn‐Gly) peptide bonds, and 2‐nitro‐5‐thiocyanobenzoic acid (NTCB) cleaves at cysteine (Cys) residues. Because the above loci are at relatively low abundance in most proteins, digestion with these agents will yield relatively long peptides. In addition, an describes CNBr cleavage of PVDF‐bound protein previously analyzed by Edman degradation. Finally, a discusses preferred methods of separating and analyzing peptide fragments generated by the chemical cleavage reactions described in the basic protocols.
Overview of Glycoconjugate Analysis
Authors: Varki Ajit , Freeze Hudson H. , Manzi Adriana E. 
Published: June 2004

AbstractWhereas DNA, RNA, and proteins are linear polymers that can usually be directly sequenced, oligosaccharides show substantially more complexity,having branching and anomeric configurations (α and β linkages). The biosynthesis of oligosaccharides, termed glycosylation, is extremely complex, is not template‐driven, varies among different cell types, and cannot be easily predicted from simple rules. This overview discusses the stereochemistry of mono‐ and oligosaccharides and provides diagrammatic representations of monosaccharides (Fisher projections and Haworth representations) and formulas for representation of oligosaccharide chains. A glossary of terms used in glycobiology is also provided.
Metabolic Radiolabeling of Animal Cell Glycoconjugates
Authors: Diaz Sandra , Varki Ajit 
Published: June 2004

AbstractUseful information about glycoconjugates can be obtained by labeling their aglycone (noncarbohydrate) portions‐‐e.g., labeling proteins with radioactive amino acids‐and then using techniques described elsewhere in this chapter to infer the presence, type, and nature of oligosaccharide chains. This unit describes metabolic labeling techniques that provide more specific information about the structure, sequence, and distribution of the sugar chains of glycoconjugates. Following metabolic labeling, the radioactive glycoconjugate of interest is isolated, individual glycosylation sites are identified and separated if necessary, and the labeled oligosaccharides are subjected to structural analysis.
Endoglycosidase and Glycoamidase Release of N‐Linked Oligosaccharides
Authors: Freeze Hudson H. 
Published: June 2004

AbstractCarbohydrate chain modifications are often used to monitor glycoprotein movement through the secretory pathway. This is because stepwise sugar‐chain processing is unidirectional and generally corresponds to the forward or anterograde movement of proteins. This unit presents a group of techniques for analysis of the general structure of carbohydrate chains on a protein and, therefore, oligosaccharide processing mileposts. It has been revised from a previous version to include endoglycosidase H, D, F2, and F3 digestions, as well as peptide:N‐glycosidase F (PNGase F), neuraminidase, endo‐β‐galactosidase, and endo‐α‐N‐acetylgalactosaminidase digestions.
Determining the Structure of Oligosaccharides N‐ and O‐Linked to Glycoproteins
Authors: Rudd Pauline M. , Dwek Raymond A. 
Published: June 2004

AbstractMany proteins involved in biological events are glycosylated. A glycoprotein consists of a mixture of glycosylation variants of a single polypeptide chain, known as glycoforms. It has become clear that a detailed understanding of the roles which glycosylation plays in the biosynthesis, transport, biological function, and degradation of a glycoprotein can only be achieved when the protein and sugar(s) are viewed as an entity. Many glycoproteins can now be modeled by combining glycan sequencing data and oligosaccharide structural information with protein structural data. Pivotal to this approach is sensitive, state‐of‐the‐art oligosaccharide sequencing technology which can give a rapid insight into the glycosylation of a glycoprotein without the need for sophisticated equipment and expertise. This unit gives a detailed introduction into the analysis of glycans, and the many figures will help the user identify which type of experiment needs to be undertaken. Methods for releasing glycans from glycoproteins are followed by protocols for labeling and purifying (by HPLC) the glycans from the rest of the components. Strategies for N‐ and O‐glycan analysis are also included.
Overview of Protein Phosphorylation
Authors: Sefton Bartholomew M. , Shenolikar Shirish 
Published: June 2004

AbstractThis overview provides a history of protein phosphorylation research and provides the reader with an understanding of how and why labeling studies are performed. The various sites of protein phosphorylation are described along with the roles of the many kinases and phosphatases that regulate phosphorylation. Methods for detecting unlabeled phosphoamino acids, including high‐voltage electrophoresis on thin‐layer cellulose acetate plates, gel‐shift assays, and the use of anti‐phosphopeptide antibodies.
Preparation and Application of Polyclonal and Monoclonal Sequence‐Specific Anti‐Phosphoamino Acid Antibodies
Authors: Sun Tong , Arlinghaus Ralph B. 
Published: June 2004

AbstractThis unit discusses the issues that must be considered in the design, production, and characterization of polyclonal and monoclonal sequence‐specific anti‐phosphoamino acid antibodies. Protocols are provided for generating and purifying such antibodies, and methods are also provided for producing useful polyclonal antibodies in a non‐purified form. Support protocols describe coupling of peptides or phosphotyrosine to a solid support for use in affinity chromatography. An example of the generation, purification, and characterization of two sequence‐specific anti‐phosphopeptide antibodies specific for different sequences of a single phosphoprotein is described. The cross‐reactivity of such antibodies, which is a common problem with anti‐peptide antibodies, is also discussed.
Assays of Protein Kinases Using Exogenous Substrates
Authors: Carter Nigel 
Published: June 2004

AbstractIn studies of the regulation of specific biochemical events by reversible phosphorylation, assaying the protein kinases themselves can often lead to significant progress in understanding the mechanistic details of a system under study. This unit describes assays for a variety of protein kinases that require different conditions to detect and measure their activities‐‐cyclic nucleotide‐dependent kinases, protein kinase C and isoforms, casein kinases, Ca2+/calmodulin‐dependent kinases, and tyrosine kinase. The unit is not meant to be a catalog of individual protein kinase assays; however, the general principles of these assays should apply to most if not all known protein kinases. In‐gel assays for specific kinase activity are also described. Support protocols describe production of crude cell lysates as a source of enzyme activity, and methods of evaluating assays, including trichloroacetic acid (TCA) precipitation to measure incorporation of radioactivity, adsorption of the labeled material onto P81 phosphocellulose paper, or electrophoretic separation and autoradiography.
Use of Protein Phosphatase Inhibitors
Authors: Weiser Douglas C. , Shenolikar Shirish 
Published: June 2004

AbstractReversible protein phosphorylation is recognized as a major mechanism regulating the physiology of plant and animal cells. Virtually every biochemical process within eukaryotic cells is controlled by the covalent modification of key regulatory proteins. This in turn dictates the cellular response to a variety of physiological and environmental stimuli; errors in signals transduced by phosphoproteins contribute to many human diseases. Thus, defining protein phosphorylation events, and specifically, the phosphoproteins involved, is crucial for obtaining a better understanding of the physiological events that distinguish normal and diseased states. Protein phosphatase inhibitors are useful when deciphering physiological events regulated by reversible protein phosphorylation but the hormonal stimuli or signaling pathways involved are not known. They are also useful in analyzing the impact of hormones and other physiological stimuli on the function of a specific phosphoprotein. This unit describes protocols for inhibiting the cellular PP1/PP2A activity with okadaic acid, microcystin‐LR, and PP2B/calcineurin and a widely utilized strategy for inhibiting protein tyrosine phosphatases.
Analysis of Protein Prenylation and Carboxyl‐Methylation
Authors: Magee Anthony I. 
Published: June 2004

AbstractThis unit describes methods for analysis of prenylation and the carboxyl‐methylation that often accompanies it. The two prenoid groups that have been found attached to proteins‐‐farnesyl (C15) and geranylgeranyl (C20)‐‐are both derived from intermediates in the isoprenoid biosynthetic pathway that utilizes mevalonic acid. In the protocols described here, radiolabeled mevalonate is used to label these intermediates in either intact cells or in vitro; the labeled intermediates then become incorporated into proteins. Alternatively, the preformed radioactive prenyl diphosphates can be used for in vitro translations, as described here. Carboxyl‐methylation of C‐terminal prenylated cysteine residues often occurs subsequent to prenylation. Methods are given for radiolabeling of the methyl group with [3H‐methyl]methionine, that is converted intracellularly intoS‐adenosylmethionine, and for radiolabeling with preformedS‐adenosyl[3H]methionine.