Current Protocols in Protein Science最新文献

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. Once the complex is purified by washing the column, intein-mediated cleavage and release of the tagless target is induced with a simple shift in buffer pH from 8.5 to 6.2. The result is a convenient and effective method for the purification of traceless and tagless target proteins, which can be used in characterization and functional studies. © 2018 by John Wiley & Sons, Inc.

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. Proteins identified by BioID are candidate interactors for the protein of interest. BioID can be applied to insoluble proteins, can identify weak and/or transient interactions, and is amenable to temporal regulation. Initially applied to mammalian cells, BioID has potential application in a variety of cell types from diverse species. © 2018 by John Wiley & Sons, Inc.

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. We also cover the use of small-scale test expression to provide an initial indication of quality and protein yield. Whereas proteins expressed at high levels can be directly scaled up, more challenging proteins may require optimization of cell lines, growth conditions, or harvest times. Scale-up and purification approaches are discussed, focusing on working with large shake cultures and use of the Wave bioreactor. © 2018 by John Wiley & Sons, Inc.

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. Here we present a chromatography-based approach for highly efficient enrichment of methylated peptides. Protocols for the of high pH SCXtip preparation and methyl-peptide purification are described in detail. Key points such as cell culture in hM-SILAC medium and protein digestion by multiple endopeptidases are also presented. This technique allows the simultaneous analysis of both lysine and arginine methylation and improved performance for methyl-arginine identification. © 2018 by John Wiley & Sons, Inc.

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. In general, when using commercial kits, satisfactory results can be readily obtained by following the manufacturer's protocols. This unit reviews commonly used fixation-based stains and provides a number of manual formulations with staining protocols for those who prefer such staining methods. © 2018 by John Wiley & Sons, Inc.

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. In contrast to antibodies derived from immunizations with detergent-solubilized protein or with protein fragments, antibodies from genetic immunization are expected to have a high capacity for binding conformational epitopes and for modulating membrane protein activity. © 2018 by John Wiley & Sons, Inc.

Procedures are described for constructing and using a microscale electrospray interface for direct infusion of samples into mass spectrometers. The sensitivity of the nanospray interface is a result of greatly reducing the flow of sample solution while preserving the analyte signal intensity. The described methodology provides a simple and robust way to analyze individual purified peptide and protein samples, i.e., samples that do not require liquid chromatography separation. Curr. Protoc. Protein Sci. 74:16.8.1-16.8.7. © 2013 by John Wiley & Sons, Inc.

The resurgence of pulsed electron paramagnetic resonance (EPR) in structural biology centers on recent improvements in distance measurements using the double electron-electron resonance (DEER) technique. This unit focuses on EPR-based distance measurements by site-directed spin labeling (SDSL) of engineered cysteine residues in soluble proteins, with HIV-1 protease used as a model. To elucidate conformational changes in proteins, experimental protocols were optimized and existing data analysis programs were employed to derive distance-distribution profiles. Experimental considerations, sample preparation, and error analysis for artifact suppression are also outlined herein. Curr. Protoc. Protein Sci. 74:17.17.1-17.17.29. © 2013 by John Wiley & Sons, Inc.

Recently, several structural genomics centers have been established and a remarkable number of three-dimensional structures of soluble proteins have been solved. For membrane proteins, the number of structures solved has been significantly trailing those for their soluble counterparts, not least because over-expression and purification of membrane proteins is a much more arduous process. By using high-throughput technologies, a large number of membrane protein targets can be screened simultaneously and a greater number of expression and purification conditions can be employed, leading to a higher probability of successfully determining the structure of membrane proteins. This unit describes the cloning, expression, and screening of membrane proteins using high-throughput methodologies developed in the laboratory. Basic Protocol 1 describes cloning of inserts into expression vectors by ligation-independent cloning. Basic Protocol 2 describes the expression and purification of the target proteins on a miniscale. Lastly, for the targets that do express on the miniscale, Basic Protocols 3 and 4 outline the methods employed for the expression and purification of targets on a midi-scale, as well as a procedure for detergent screening and identification of detergent(s) in which the target protein is stable. Curr. Protoc. Protein Sci. 74:29.6.1-29.6.34. © 2013 by John Wiley & Sons, Inc.

Antibody arrays provide a valuable method for obtaining multiple protein measurements from small volumes of biological samples. Antibody arrays can be designed to target not only core protein abundances (relative or absolute abundances, depending on the availability of standards for calibration), but also posttranslational modifications, provided antibodies or other affinity reagents are available to detect them. Glycosylation is a common modification that has important and diverse functions in both normal and disease biology. Significant progress has been made in developing methods for measuring glycan levels on multiple specific proteins using antibody arrays and glycan-binding reagents. This unit describes practical approaches for developing, optimizing, and using antibody array assays to determine both protein abundance and glycosylation state. Low-volume arrays can be used to reduce sample consumption, and a new way to improve the binding strength of particular glycan-binding reagents through multimerization is discussed. These methods can be useful for a wide range of biological studies in which glycosylation may change and/or affect protein function. Curr. Protoc. Protein Sci. 73:27.6.1-27.6.16. © 2013 by John Wiley & Sons, Inc.