high-throughput-quantitative-expression-screening-purification

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in <em>E. coli</em>

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Aix-Marseille Université

Munc18-1 and Syntaxin1 are essential proteins for SNARE-mediated neurotransmission. Munc18-1 participates in synaptic vesicle fusion via dual roles: as a docking/chaperone protein by binding closed Syntaxin1, and as a fusion protein that binds SNARE complexes in a Syntaxin1 N-peptide dependent manner. The two roles are associated with a closed-open Syntaxin1 conformational transition. Here, we show that Syntaxin N-peptide binding to Munc18-1 is not highly selective, suggesting that other parts of the SNARE complex are involved in binding to Munc18-1. We also find that Syntaxin1, with an N peptide and a physically anchored C terminus, binds to Munc18-1 and that this complex can participate in SNARE complex formation. We report a Munc18-1-N-peptide crystal structure that, together with other data, reveals how Munc18-1 might transit from a conformation that binds closed Syntaxin1 to one that may be compatible with binding open Syntaxin1 and SNARE complexes. Our results suggest the possibility that structural transitions occur in both Munc18-1 and Syntaxin1 during their binary interaction. We hypothesize that Munc18-1 domain 3a undergoes a conformational change that may allow coiled-coil interactions with SNARE complexes.

Possible roles for Munc18-1 domain 3a and Syntaxin1 N-peptide and C-terminal anchor in SNARE complex formation.

Acid-sensing ion channel 1a (ASIC1a) is a primary acid sensor in the peripheral and central nervous system. It has been implicated as a novel therapeutic target for a broad range of pathophysiological conditions including pain, ischemic stroke, depression, and autoimmune diseases such as multiple sclerosis. The only known selective blocker of ASIC1a is π-TRTX-Pc1a (PcTx1), a disulfide-rich 40-residue peptide isolated from spider venom. π-TRTX-Pc1a is an effective analgesic in rodent models of acute pain and it provides neuroprotection in a mouse model of ischemic stroke. Thus, understanding the molecular basis of the π-TRTX-Pc1a-ASIC1a interaction should facilitate development of therapeutically useful ASIC1a blockers. We therefore developed an efficient bacterial expression system to produce a panel of π-TRTX-Pc1a mutants for probing structure-activity relationships as well as isotopically labeled toxin for determination of its solution structure and dynamics. We demonstrate that the toxin pharmacophore resides in a β-hairpin loop that was revealed to be mobile over a wide range of time scales using molecular dynamics simulations in combination with NMR spin relaxation and relaxation dispersion measurements. The toxin-receptor interaction was modeled by in silico docking of the toxin structure onto a homology model of rat ASIC1a in a restraints-driven approach that was designed to take account of the dynamics of the toxin pharmacophore and the consequent remodeling of side-chain conformations upon receptor binding. The resulting model reveals new insights into the mechanism of action of π-TRTX-Pc1a and provides an experimentally validated template for the rational design of therapeutically useful π-TRTX-Pc1a mimetics.

A dynamic pharmacophore drives the interaction between Psalmotoxin-1 and the putative drug target acid-sensing ion channel 1a.

Spiders are the most successful venomous animals and the most abundant terrestrial predators. Their remarkable success is due in large part to their ingenious exploitation of silk and the evolution of pharmacologically complex venoms that ensure rapid subjugation of prey. Most spider venoms are dominated by disulfide-rich peptides that typically have high affinity and specificity for particular subtypes of ion channels and receptors. Spider venoms are conservatively predicted to contain more than 10 million bioactive peptides, making them a valuable resource for drug discovery. Here we review the structure and pharmacology of spider-venom peptides that are being used as leads for the development of therapeutics against a wide range of pathophysiological conditions including cardiovascular disorders, chronic pain, inflammation, and erectile dysfunction.

Spider-venom peptides as therapeutics.

Disulfide-rich proteins or DRPs are versatile bioactive compounds that encompass a wide variety of pharmacological, therapeutic, and/or biotechnological applications. Still, the production of DRPs in sufficient quantities is a major bottleneck for their complete structural or functional characterization. Recombinant expression of such small proteins containing multiple disulfide bonds in the bacteria E. coli is considered difficult and general methods and protocols, particularly on a high throughput scale, are limited.

High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli.

Disulfide-rich peptides are the dominant component of most animal venoms. These peptides have received much attention as leads for the development of novel therapeutic agents and bioinsecticides because they target a wide range of neuronal receptors and ion channels with a high degree of potency and selectivity. In addition, their rigid disulfide framework makes them particularly well suited for addressing the crucial issue of in vivo stability. Structural and functional characterization of these peptides necessitates the development of a robust, reliable expression system that maintains their native disulfide framework. The bacterium Escherichia coli has long been used for economical production of recombinant proteins. However, the expression of functional disulfide-rich proteins in the reducing environment of the E. coli cytoplasm presents a significant challenge. Thus, we present here an optimised protocol for the expression of disulfide-rich venom peptides in the periplasm of E. coli, which is where the endogenous machinery for production of disulfide-bonds is located. The parameters that have been investigated include choice of media, induction conditions, lysis methods, methods of fusion protein and peptide purification, and sample preparation for NMR studies. After each section a recommendation is made for conditions to use. We demonstrate the use of this method for the production of venom peptides ranging in size from 2 to 8 kDa and containing 2-6 disulfide bonds.

Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli.

The protocols outlined in this chapter allow for the small-scale test expression of a single or multiple proteins concurrently using several expression conditions to identify optimal strategies for producing soluble, stable proteins. The protocols can be performed manually without the need for specialized equipment, or can be translated to robotic platforms. The high-throughput protocols begin with transformation in a 96-well format, followed by small-scale test expression using auto-induction medium in a 24-well format, finishing with purification in a 96-well format. Even from such a small scale, there is the potential to use the purified proteins for characterization in pilot studies, for sensitive micro-assays, or for the quick detection of and differentiation of the expected size and oxidation state of the protein by mass spectrometry.

Article	Year
Possible roles for Munc18-1 domain 3a and Syntaxin1 N-peptide and C-terminal anchor in SNARE complex formation. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 21193638	2011
A dynamic pharmacophore drives the interaction between Psalmotoxin-1 and the putative drug target acid-sensing ion channel 1a. Molecular pharmacology\| PubMed ID: 21825095	2011
Spider-venom peptides as therapeutics. Toxins\| PubMed ID: 22069579	2010
High throughput screening identifies disulfide isomerase DsbC as a very efficient partner for recombinant expression of small disulfide-rich proteins in E. coli. Microbial cell factories\| PubMed ID: 23607455	2013
Production of recombinant disulfide-rich venom peptides for structural and functional analysis via expression in the periplasm of E. coli. PloS one\| PubMed ID: 23667680	2013
High-throughput expression screening and purification of recombinant proteins in E. coli. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 24203323	2014

Natalie J. Saez

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