The Expanding Toolkit of Translating Ribosome Affinity Purification.

Translating ribosome affinity purification is a method initially developed for profiling mRNA from genetically defined cell types in complex tissues. It has been applied both to identify target molecules in cell types that are important for controlling a variety of behaviors in the brain, and to understand the molecular consequences on those cells due to experimental manipulations, ranging from drugs of abuse to disease-causing mutations. Since its inception, a variety of methodological advances are opening new avenues of investigation. These advances include a variety of new methods for targeting cells for translating ribosome affinity purification by features such as their projections or activity, additional tags and mouse reagents increasing the flexibility of the system, and new modifications of the method specifically focused on studying the regulation of translation. The latter includes methods to assess cell type-specific regulation of translation in specific subcellular compartments. Here, I provide a summary of these recent advances and resources, highlighting both new experimental opportunities and areas for future technical development.

The growing impact of lyophilized cell-free protein expression systems.

Recently reported shelf-stable, on-demand protein synthesis platforms are enabling new possibilities in biotherapeutics, biosensing, biocatalysis, and high throughput protein expression. Lyophilized cell-free protein expression systems not only overcome cold-storage limitations, but also enable stockpiling for on-demand synthesis and completely sterilize the protein synthesis platform. Recently reported high-yield synthesis of cytotoxic protein Onconase from lyophilized E. coli extract preparations demonstrates the utility of lyophilized cell-free protein expression and its potential for creating on-demand biotherapeutics, vaccines, biosensors, biocatalysts, and high throughput protein synthesis.

New frontiers in gut nutrient sensor research: luminal glutamate-sensing cells in rat gastric mucosa.

Dietary free glutamate is known to elicit umami, one of the five basic tastes perceived via the specific taste sensor cells on the tongue. Recent studies suggest the specific glutamate sensors exist in the gastric mucosa and contribute to the regulation of gastrointestinal functions, yet the precise mechanism remains still unknown. We established the method to enrich various cell fractions from the isolated rat gastric mucosa and characterized the expression of putative glutamate sensors using such cell fractions. The gastric mucosal cell fractions such as surface mucous, parietal, chief, and endocrine cells were successfully prepared by mucosal protease digestion, elutriation, and gradient centrifugation. The characteristics of these cells were confirmed by real-time RT-PCR using the respective cell-specific markers. Parietal cell fraction exclusively expressed putative umami receptor molecules such as T1R1 and mGluR1 compared to other fractions, although the degree of expression was low. In contrast, the representative taste cell specific markers such as PLCbeta2 and TRPM5 were specifically expressed in the smaller endocrine cell fraction. Both parietal and smaller endocrine cell fractions also positively expressed some mGluR subtypes. The chief-cell fraction less expressed T1R1 and mGluR1. These results suggest that multiple glutamate sensors, probably different mechanisms from taste buds, contribute to the glutamate sensing in the gastric mucosa.

Advances in preparation of biological extracts for protein purification.

There are a variety of reliable methods for cellular disintegration and extraction of proteins ranging from enzymatic digestion and osmotic shock to ultrasonication, and pressure disruption. Each method has inherent advantages and disadvantages. Generally vigorous mechanical treatments reduce extract viscosity but can result in the inactivation of labile proteins by heat or oxidation, while gentle treatments may not release the target protein from the cells, and resulting extracts are extremely viscous. Depending on the cell type selected as the source for target protein expression, cellular extracts contain large amounts of nucleic acid, ribosomal material, lipids, dispersed cell wall polysaccharide, carbohydrates, chitin, small molecules, and thousands of unwanted proteins. Isolation and recovery of a single protein from this complex mixture of macromolecules presents considerable challenges. The first and possibly most important of these challenges is generation of a cellular extract that can be efficiently manipulated in downstream processes without inactivation or degradation of labile protein targets. Cell disruption techniques must rapidly and efficiently lyse cells to extract proteins with minimal proteolysis or oxidation while reducing extract viscosity caused by cell debris and genomic DNA contamination. Advanced bioprocessing equipment and reagents have been developed over the past twenty years to complement established disruption procedures and accomplish these tasks with even greater success. This chapter will summarize these advances and describe detailed protocols for some of the most popular methods for protein extraction.

Cell lysis methods for high-throughput screening or miniaturized assays.

The widely and routinely used cell lysis techniques are still being evolved to address the technical needs of micro-scale sample processing, for example in high-throughput screening or miniaturized assays. This review focuses on several recently emerged mechanical, physical and biological methods designed for lysing cells on small scales for screening or analysis.

Complementary methods to assist subcellular fractionation in organellar proteomics.

Organellar proteomics aims to describe the full complement of proteins of subcellular structures and organelles. When compared with whole-cell or whole-tissue proteomes, the more focused results from subcellular proteomic studies have yielded relatively simpler datasets from which biologically relevant information can be more easily extracted. In every proteomic study, the quality and purity of the biological sample to be investigated is of the utmost importance for a successful analysis. In organellar proteomics, one of the most crucial steps in sample preparation is the initial subcellular fractionation procedure by which the enriched preparation of the sought-after organelle is obtained. In nearly all available organellar proteomic studies, the method of choice relies on one or several rounds of density-based gradient centrifugation. Although this method has been recognized for decades as yielding relatively pure preparations of organelles, recent technological advances in protein separation and identification can now reveal even minute amounts of contamination, which in turn can greatly complicate data interpretation. The scope of this review focuses on recently published innovative complementary or alternative methods to perform subcellular fractionation, which can further refine the way in which sample preparation is accomplished in organellar proteomics.