Current Protocols in Protein Science最新文献

Characterization of the higher-order structure (HOS) of protein therapeutics, and in particular of monoclonal antibodies, by 2D ¹H-¹³C methyl correlated NMR has been demonstrated as precise and robust. Such characterization can be greatly enhanced when collections of spectra are analyzed using multivariate approaches such as principal component analysis (PCA), allowing for the detection and identification of small structural differences in drug substance that may otherwise fall below the limit of detection of conventional spectral analysis. A major limitation to this approach is the presence of aliphatic signals from formulation or excipient components, which result in spectral interference with the protein signal of interest; however, the recently described Selective Excipient Reduction and Removal (SIERRA) filter greatly reduces this issue. Here we will outline how basic 2D ¹H-¹³C methyl−correlated NMR may be combined with the SIERRA approach to collect ‘clean’ NMR spectra of formulated monoclonal antibody therapeutics (i.e., drug substance spectra free of interfering component signals), and how series of such spectra may be used for HOS characterization by direct PCA of the series spectral matrix. © 2020 U.S. Government.

Basic Protocol 1: NMR data acquisition

Basic Protocol 2: Full spectral matrix data processing and analysis

Support Protocol: Data visualization and cluster analysis

Heterologous expression of exogenous proteases in Escherichia coli often results in the formation of insoluble inclusion bodies. When sequestered into inclusion bodies, the functionality of the proteases is minimized. To be characterized structurally and functionally, however, proteases must be obtained in their native conformation. HIV protease is readily expressed as inclusion bodies, but must be recovered from the inclusion bodies. This protocol describes an efficient method for recovering HIV protease from inclusion bodies, as well as refolding and purifying the protein. HIV protease–containing inclusion bodies are treated with 8 M urea and purified via cation-exchange chromatography. Subsequent refolding by buffer exchange via dialysis and further purification by anion-exchange chromatography produces highly pure HIV protease that is functionally active. © 2020 by John Wiley & Sons, Inc.

Basic Protocol: Recovery, refolding, and purification of HIV protease from inclusion bodies

Support Protocol 1: Expression and extraction of inclusion bodies containing HIV protease expressed in Escherichia coli

Support Protocol 2: Determination of the active site concentration of HIV protease via isothermal titration calorimetry

Pichia pastoris is a eukaryotic microorganism reputed for its ability to mass-produce recombinant proteins, including integral membrane proteins, for various applications. This article details a series of protocols that progress towards the production of integral membrane proteins, their extraction and purification in the presence of detergents, and their eventual reconstitution in lipid nanoparticles. These basic procedures can be further optimized to provide integral membrane protein samples that are compatible with a number of structural and/or functional investigations at the molecular level. Each protocol provides general guidelines, technical hints, and specific recommendations, and is illustrated with case studies corresponding to several representative mammalian proteins. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Production of membrane proteins in a P. pastoris recombinant clone using methanol induction

Basic Protocol 2: Preparation of whole-membrane fractions

Alternate Protocol 1: Preparation of yeast protoplasts

Basic Protocol 3: Extraction of membrane proteins from whole-membrane fractions

Basic Protocol 4: Purification of membrane proteins

Alternate Protocol 2: Purification of membrane proteins from yeast protoplasts

Alternate Protocol 3: Simultaneous protoplast preparation and membrane solubilization for purification of membrane proteins

Basic Protocol 5: Reconstitution of detergent-purified membrane proteins in lipid nanoparticles

Lectin is a biomolecule that recognizes a specific part of glycans and, thus, has been used widely as a probe for glycoprotein analysis. Owing to the wide repertoire in nature combined with the recent two decades of advances in microarray technology, the multiplexed use of lectins has been widely used for glycan profiling of endogenous proteins. Because protein glycosylation is recognized as being biologically important and is expected to be a reliable disease marker, lectin microarray analysis with highly sensitive detection has been used to discover disease-relevant glycosylation alterations. However, the conventional system is limited to research purposes; thus, its implementation in clinical settings is warranted. Here, we provide an automatic glycan profiling method using GlycoBIST. A unique array format is used for 10-plexed lectin–glycoprotein interaction analysis on 1-mm-sized beads, which are arranged vertically in a capillary-shaped plastic tip. Using a one-boxed autopipetting machine, the whole process (including interaction, washing, and detection) is performed automatically and serially, resulting in reproducible measurements. In this article, a typical method for glycan profiling of a purified glycoprotein and the fabrication of GlycoBIST tips is explained. © 2020 by John Wiley & Sons, Inc.

Basic Protocol 1: Fabrication of a GlycoBIST tip

Basic Protocol 2: Automatic profiling of a target glycoprotein using GlycoBIST

The water-proton signal, overwhelmingly considered a nuisance in nuclear magnetic resonance spectroscopy, is advantageously used as a tool to assess protein concentration and to detect protein aggregates in aqueous solutions. The protocols in this article describe use of the water-proton transverse relaxation rate to determine concentration and aggregate content in protein solutions. Detailed recommendations and description of the parameter settings and data processing ensure successful implementation of this technique, even by a user with limited experience in magnetic resonance relaxometry. All measurements are done noninvasively, in a sealed container, without sampling or otherwise aliquoting the solution. The magnetic resonance relaxometry approach offered in this article could be advantageous for analysis of biologics formulations or when use of conventional analytical techniques is not possible. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Nuclear magnetic resonance (NMR) relaxometry to measure protein concentration

Basic Protocol 2: NMR relaxometry to measure protein aggregation

Analysis of RNA structuromes provides new insights into cellular processes, enabling systems biology and biotechnology researchers to calculate promoter and terminator strengths and to directly observe how differing circuit states impact host gene expression and the burdens imposed by the circuits. Such analysis, however, is crucially dependent on the availability of highly pure, intact RNA isolated from fresh or frozen cell cultures. RNA extraction from the yeast Pichia pastoris requires specific pretreatment steps to ensure the reproducibility of downstream applications, but current methods and extraction kits are generally adapted for the conventional yeast Saccharomyces cerevisiae, which has a different cell wall composition. We therefore set out to compare the efficacy of two different RNA isolation methods when applied to P. pastoris: (i) phenol/chloroform extraction and (ii) silica spin-column absorption. We compared the yield, integrity, and purity of the resulting isolated RNA from the two methods (using two different types of commercial columns for silica spin-column absorption) and further optimized them through variations in the pretreatment steps. We also assessed two different methods of cell lysis: enzyme catalytic disruption using lyticase and mechanical disruption using acid-washed glass-beads in a TissueLyser. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: RNA isolation with phenol/chloroform extraction: monophasic lysis reagent

Alternate Protocol 1: RNA isolation with silica-spin column absorption: High Pure RNA Isolation Kit (Roche Life Science)

Alternate Protocol 2: RNA isolation with silica-spin column absorption: RNeasy Mini Kit (Qiagen)

Lipid nanodiscs provide a native-like lipid environment for membrane proteins, and they have become a valuable platform for the study of membrane biophysics. A range of biophysical and biochemical analyses are enabled when membrane proteins are captured in lipid nanodiscs. Two parameters that can be controlled when capturing membrane proteins in lipid nanodiscs are the radius, and hence the surface area of the lipid surface, and the composition of the lipid bilayer. Despite their emergence as a versatile tool, most studies with lipid nanodiscs in the literature have focused on nanodiscs of a single radius with a single lipid. In light of the complexity of biological membranes, it is likely that nanodiscs with multiple membrane components would be more sophisticated models for membrane research. It is possible to prepare nanodiscs with more complex lipid mixtures to probe the effects of lipid composition on several aspects of membrane biochemistry. Detailed protocols are described here for the preparation of nanodiscs with mixtures of phospholipids, incorporation of cholesterol, and incorporation of a spectroscopic lipid probe. These protocols provide starting points for the construction of nanodiscs with more physiological membrane compositions or with useful biophysical probes. © 2019 by John Wiley & Sons, Inc.

Basic Protocol 1: Assembly of mixed lipid nanodiscs

Basic Protocol 2: Assembly of nanodiscs with cholesterol

Basic Protocol 3: Incorporation of laurdan into nanodiscs for membrane fluidity measurements