Current Protocols in Protein Science最新文献

Actin is one of the most abundant proteins in the cytoplasm of eukaryotic cells and plays important roles in a variety of cellular functions. However, it has been difficult to produce actin in substantial amounts using bacterial expression systems. In this article, a new method is described for the production of recombinant actin in bacterial cells. Human β-actin (His-tagged) can be expressed using a cold shock vector, pCold, in a bacterial expression system and then separated with a Ni-chelating resin, followed by a polymerization/depolymerization cycle or column chromatography with the Ni-chelating resin. The purified recombinant β-actin shows normal polymerization ability compared with commercially available β-actin purified from human platelets. This article also describes the preparation of mutant actin(G168R). This purified mutant exhibits impaired polymerization ability. The system and procedures described here will provide a useful method for the production of actin isoforms and their mutants. © 2018 by John Wiley & Sons, Inc.

This updated unit compares three methods to acquire Förster Resonance Energy Transfer (FRET) data in living cells using a confocal microscope: Acceptor photobleaching, Acceptor-sensitized emission FRET, and Donor fluorescence lifetime imaging. Detailed protocols for live cell husbandry, image acquisition, and data analysis are provided. In addition to providing instructions for manufacturer's analysis tool sets, we provide an easy-to-use, MATLAB-based code to calculate FRET efficiency from data obtained using the Acceptor photobleaching or Acceptor-sensitized emission method, which can be freely downloaded. © 2018 by John Wiley & Sons, Inc.

Understanding protein-protein interactions (PPIs) in a cell is essential for learning protein functions, pathways, and mechanism of diseases. PPIs are also important targets for developing drugs. Experimental methods, both small-scale and large-scale, have identified PPIs in several model organisms. However, results cover only a part of PPIs of organisms; moreover, there are many organisms whose PPIs have not yet been investigated. To complement experimental methods, many computational methods have been developed that predict PPIs from various characteristics of proteins. Here we provide an overview of literature reports to classify computational PPI prediction methods that consider different features of proteins, including protein sequence, genomes, protein structure, function, PPI network topology, and those which integrate multiple methods. © 2018 by John Wiley & Sons, Inc.

Studies aiming at heterologous expression of highly hydrophobic proteins, such as outer membrane proteins in general and peptidoglycan-associated lipoprotein (PAL) in particular, are not trivial due to difficulties in obtaining recombinant protein in a soluble state, which is desired because it allows purification by traditional chromatographic methods. PAL is associated with the integrity of the cellular envelope in Gram-negative bacteria and interacts strongly with the peptidoglycan layer. However, it is incorporated into inclusion bodies in studies focusing on its heterologous production. This protocol describes an efficient protein refolding method to solubilize and purify a recombinant PAL. Initially, recombinant PAL–enriched inclusion bodies obtained after the induction of PAL expression in Escherichia coli are treated with 8 M urea and then undergo buffer exchange via dialysis. Afterward, the soluble, recombinant PAL is purified using standard chromatographic methods. © 2018 by John Wiley & Sons, Inc.

This unit describes production of a bacterial thermophilic xylanase enzyme in an industrially exploited filamentous fungus, Trichoderma reesei. Successful expression of a gene of interest in a heterologous host involves front-end design of the expression constructs using bioinformatics tools, making the constructs in the laboratory, and introducing them into the expression host. This is followed by synthesis and characterization of the gene product on a laboratory scale and optimization of the cultivation parameters in a controlled, scaled-up fermentation. The thermophilic xylanase B (XynB) enzyme from the bacterium Dictyoglomus thermophilum discussed here can be easily purified by heat-precipitation from the culture supernatant of the mesophilic host. A functional XynB can also be produced in Escherichia coli, but at a lower yield compared to that obtained in T. reesei. The protocol provided here can be adapted to various other proteins and filamentous fungal hosts. © 2018 by John Wiley & Sons, Inc.

Filamentous fungi are lower eukaryotes increasingly used for expression of foreign proteins ranging from industrial enzymes originating from other fungi and bacteria to proteins of mammalian origin, such as antibodies and growth factors. Their strengths include an excellent capacity for protein secretion and their eukaryotic protein processing machinery. Proteins secreted from filamentous fungi are modified in the secretory pathway, with folding, proteolytic processing, and addition of glycans being the main modifications. Unlike from many other expression systems, however, plasmids and host strains for expression of gene products in filamentous fungi are not readily available commercially, and the expression system must thus be stitched together in the laboratory. In this overview, the key elements of fungal expression systems are discussed from a practical point of view and with a view towards the future. The principles and considerations presented here can be applied to a range of filamentous fungi. © 2018 by John Wiley & Sons, Inc.

Histone N termini undergo diverse post-translational modifications that significantly extend the information potential of the genetic code. Moreover, these modifications mark specific chromatin regions, modulating epigenetic control, lineage commitment, and overall function of chromosomes. It is widely accepted that histone modifications affect chromatin function, but the exact mechanisms by which modifications on histone tails and specific combinations of modifications are generated, and how they cross-talk with one another, are still enigmatic. Mass spectrometry is the gold-standard method for analyzing histone modifications, as it allows the quantification of modifications and combinations. This unit describes how high-resolution mass spectrometry can be used to study histone post-translational modifications. © 2018 by John Wiley & Sons, Inc.

Carbamylation corresponds to the non-enzymatic binding of isocyanic acid to protein amino groups and participates in protein molecular aging, characterized by the alteration of their structural and functional properties. Carbamylated proteins exert deleterious effects in vivo and are involved in the progression of various diseases, including atherosclerosis and chronic kidney disease. Therefore, there is a growing interest to evaluate the carbamylation rate of blood or tissue proteins, since carbamylation-derived products (CDPs) constitute valuable biomarkers in these contexts. Homocitrulline, formed by isocyanic acid covalently attaches to the ε-NH₂ group of lysine residue side chain, is the most characteristic CDP. Sensitive and specific quantification of homocitrulline requires mass spectrometry-based methods. This unit describes a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of homocitrulline, with special emphasis on pre-analytical steps that allow quantification of total or protein-bound homocitrulline in serum or tissue samples. © 2018 by John Wiley & Sons, Inc.

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. We describe advances made to the BEVS to allow the rapid and easy production of recombinant viruses and developments to improve protein yield. We finish by describing the application of recombinant BacMam as vectors for the delivery of genes into mammalian and human cells. © 2018 by John Wiley & Sons, Inc.