Miniaturisation of high-throughput plasmid DNA library preparation for next-generation sequencing using multifactorial optimisation.

High-throughput preparation of plasmid DNA libraries for next-generation sequencing (NGS) is an important capability for molecular biology laboratories. In particular, it is an essential quality control (QC) check when large numbers of plasmid variants are being generated. Here, we describe the use of the Design of Experiments (DOE) methodology to optimise the miniaturised preparation of plasmid DNA libraries for NGS, using the Illumina® Nextera XT technology and the Labcyte Echo® acoustic liquid dispensing system. Furthermore, we describe methods which can be implemented as a QC check for identifying the presence of genomic DNA (gDNA) in plasmid DNA samples and the subsequent shearing of the gDNA, which otherwise prevents the acoustic transfer of plasmid DNA. This workflow enables the preparation of plasmid DNA libraries which yield high-quality sequencing data.

Designing experiments for high-throughput protein expression.

The advent of high-throughput protein production and the vast amount of data it is capable of generating has created both new opportunities and problems. Automation and miniaturization allow experimentation to be performed more efficiently, justifying the cost involved in establishing a high-throughput platform. These changes have also magnified the need for effective statistical methods to identify trends and relationships in the data. The application of quantitative management tools to this process provides the means of ensuring maximum efficiency and productivity.

E. coli and insect cell expression, automated purification and quantitative analysis.

The production of recombinant proteins usually involves the exploration of a wide variety of expression and purification methodologies in the pursuit of a strategy tailored to a particular protein. The methods applied are reliant on exploiting individual differences between expression systems or the variations in specific protein properties. These bespoke strategies have not lent themselves to high-throughput methodologies. Ultimately the development of robust generic methods capable of simplifying and stabilizing the process, allowing automation, was necessary to increase throughput. This chapter describes a series of high-throughput methods used to express, purify, and quantify recombinant protein produced in E. coli or insect cells.

Development of a protease production platform for structure-based drug design.

Structure-based drug design (SBDD) has played an integral role in the development of highly specific, potent protease inhibitors resulting in a number of drugs in clinical trials and on the market. Possessing biochemical assays and structural information on both the target protease and homologous family members helps ensure compound selectivity. We have redesigned the path from clone to protein eliminating many of the traditional bottlenecks associated with protein production to ensure a constant supply to feed many diverse protease drug discovery programs. The process was initiated with the design of a multi-system vector, capable of expression in both eukaryotic and prokaryotic hosts; this vector also facilitated high-throughput cloning, expression and purification. When combined into an expression screen, supplemented with salvage screens for detergent extraction and refolding, a route for protein production was established rapidly. Using this process-orientated approach we have successfully expressed and purified all mechanistic classes of active human and viral proteases for enzymatic assays and crystallization studies. While exploiting recent developments in high-throughput biochemistry, we still employ classical biophysical techniques such as light-scattering and analytical ultracentrifugation, to ensure the highest quality protein enters crystallization trials. We have drawn on examples from our own research programs to illustrate how these strategies have been successfully used in the production of proteases for SBDD.

Screening factors effecting a response in soluble protein expression: formalized approach using design of experiments.

We have integrated high-throughput expression and purification with quantitative protein analysis to identify factors influencing protein production. Application of high-throughput expression and purification, combined with automated gel capillary electrophoresis, allowed the quantitative analysis of multiple expression variables in a single experiment. An experimental design utilizing multiple factorial screens was employed to identify single factors and interactions having a significant impact on expression. As a test case, expression of the nonstructural protein NS3 from different hepatitis C virus genotypes (1b, 2a, and 3a) was examined in Escherichia coli. The 1b genotype of NS3 produced the highest level of expression, which was then further optimized using response surface modeling to give a four-fold increase in soluble protein levels. The quantitative and statistical approach presented has the capability of rapidly identifying interactions among experimental variables, leading to more reliable prediction of protein expression. We propose that this technique has universal application in the production of recombinant proteins, providing a powerful tool for the optimization of protein expression.

High-throughput screening for soluble recombinant expressed kinases in Escherichia coli and insect cells.

We have constructed a dual expression vector for the production of recombinant proteins in both Escherichia coli and insect cells. In this vector, the baculoviral polyhedrin promoter was positioned upstream of the bacteriophage T7 promoter and the lac operator. This vector, designated pBEV, was specifically designed to exploit the advantages that both hosts would provide. This vector also facilitates one-stop cloning, thereby simplifying the expression process for automation, and the development of a high-throughput method for protein expression. Utilizing the multi-system vector pBEV, a high-throughput process was developed with expression in deep-well blocks and purification in micro-titer plates enabling the identification of expression and solubility in both E. coli and insect cells. In this study, using pBEV, we have successfully expressed and purified multiple human kinases produced in E. coli and insect cells. Our results validate expression screening as a strategy to rapidly triage proteins identifying the optimum expression system and conditions for production.

Short interfering RNA-mediated gene targeting in the zebrafish.

Short interfering RNAs (siRNAs) have proved to be a useful tool in studying gene function in plants, invertebrates and mammalian systems. Here we report the use of siRNAs for targeting the zebrafish dystrophin gene. This study demonstrates the efficacy of siRNA-based gene silencing in this vertebrate model species, and illustrates the potential of this approach for determining the roles of multiple protein products expressed by a single gene during the early stages of development. In addition this study illustrates the usefulness of zebrafish as a model for muscle disease, and highlights the potential of siRNA-based gene targeting for disease analysis in this model organism.

Sarcoglycans of the zebrafish: orthology and localization to the sarcolemma and myosepta of muscle.

The zebrafish is an established model of vertebrate development and is also receiving increasing attention in terms of human disease modelling. In order to provide experimental support to realize this modelling potential, we report here the identification of apparent orthologues of many critical members of the dystrophin-associated glycoprotein complex (DGC) that have been implicated in a diverse range of neuromuscular disorders. In addition, immunohistochemical studies show the localization of the DGC to the sarcolemma of adult zebrafish muscle and in particular the myosepta. Together, these data suggest that the DGC in adult zebrafish may play a highly conserved functional role in muscle architecture that, when disrupted, could offer insight into human neuromuscular disease processes.

Active-site residues of Escherichia coli DNA gyrase required in coupling ATP hydrolysis to DNA supercoiling and amino acid substitutions leading to novobiocin resistance.

DNA gyrase is a bacterial type II topoisomerase which couples the free energy of ATP hydrolysis to the introduction of negative supercoils into DNA. Amino acids in proximity to bound nonhydrolyzable ATP analog (AMP. PNP) or novobiocin in the gyrase B (GyrB) subunit crystal structures were examined for their roles in enzyme function and novobiocin resistance by site-directed mutagenesis. Purified Escherichia coli GyrB mutant proteins were complexed with the gyrase A subunit to form the functional A(2)B(2) gyrase enzyme. Mutant proteins with alanine substitutions at residues E42, N46, E50, D73, R76, G77, and I78 had reduced or no detectable ATPase activity, indicating a role for these residues in ATP hydrolysis. Interestingly, GyrB proteins with P79A and K103A substitutions retained significant levels of ATPase activity yet demonstrated no DNA supercoiling activity, even with 40-fold more enzyme than the wild-type enzyme, suggesting that these amino acid side chains have a role in the coupling of the two activities. All enzymes relaxed supercoiled DNA to the same extent as the wild-type enzyme did, implying that only ATP-dependent reactions were affected. Mutant genes were examined in vivo for their abilities to complement a temperature-sensitive E. coli gyrB mutant, and the activities correlated well with the in vitro activities. We show that the known R136 novobiocin resistance mutations bestow a significant loss of inhibitor potency in the ATPase assay. Four new residues (D73, G77, I78, and T165) that, when changed to the appropriate amino acid, result in both significant levels of novobiocin resistance and maintain in vivo function were identified in E. coli.

High-throughput protein expression for the post-genomic era.

In the past, protein expression has been perceived as the principle bottleneck in protein characterization and structure determination. The challenge now is to rapidly express large numbers of genes in the search for new drug targets and therapeutic proteins encoded by the human genome. In this competitive environment, several high-throughput expression strategies for protein production are being used to industrialize the process of protein expression.