SLiMPrints: conservation-based discovery of functional motif fingerprints in intrinsically disordered protein regions

Large portions of higher eukaryotic proteomes are intrinsically disordered, and abundant evidence suggests that these unstructured regions of proteins are rich in regulatory interaction interfaces. A major class of disordered interaction interfaces are the compact and degenerate modules known as short linear motifs (SLiMs). As a result of the difficulties associated with the experimental identification and validation of SLiMs, our understanding of these modules is limited, advocating the use of computational methods to focus experimental discovery. This article evaluates the use of evolutionary conservation as a discriminatory technique for motif discovery. A statistical framework is introduced to assess the significance of relatively conserved residues, quantifying the likelihood a residue will have a particular level of conservation given the conservation of the surrounding residues. The framework is expanded to assess the significance of groupings of conserved residues, a metric that forms the basis of SLiMPrints (short linear motif fingerprints), a de novo motif discovery tool. SLiMPrints identifies relatively overconstrained proximal groupings of residues within intrinsically disordered regions, indicative of putatively functional motifs. Finally, the human proteome is analysed to create a set of highly conserved putative motif instances, including a novel site on translation initiation factor eIF2A that may regulate translation through binding of eIF4E.     

High‐throughput process development of purification alternatives for the protein avidin

With an increased number of applications in the field of the avidin‐biotin technology, the resulting demand for highly‐purified protein avidin has drawn our attention to the purification process of avidin that naturally occurs in chicken egg white. The high‐throughput process development (HTPD) methodology was exploited, in order to evaluate purification process alternatives to commonly used ion‐exchange chromatography. In a high‐throughput format, process parameters for aqueous two‐phase extraction, selective precipitation with salts and polyethylene glycol, and hydrophobic interaction and mixed‐mode column chromatography experiments were performed. The HTPD strategy was complemented by a high‐throughput tandem high‐performance liquid chromatography assay for protein quantification. Suitable conditions for the separation of avidin from the major impurities ovalbumin, ovomucoid, ovotransferrin, and lysozyme were identified in the screening experiments. By combination of polyethylene glycol precipitation with subsequent resolubilization and separation in a polyethylene glycol/sulfate/sodium chloride two‐phase system an avidin purity of 77% was obtained with a yield >90% while at the same time achieving a significant reduction of the process volume. The two‐phase extraction and precipitation results were largely confirmed in larger scale with scale‐up factors of 230 and 133, respectively. Seamless processing of the avidin enriched bottom phase was found feasible by using mixed‐mode chromatography. By gradient elution a final avidin purity of at least 97% and yield >90% was obtained in the elution pool. The presented identification of a new and beneficial alternative for the purification of the high value protein thus represents a successful implementation of HTPD for an industrially relevant purification task. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:957–973, 2015     

High‐throughput species identification: from DNA isolation to bioinformatics

Ichthyoplankton collections provide a valuable means to study fish life histories. However, these collections are greatly underutilized, as larval fishes are frequently not identified to species due to their small size and limited morphological development. Currently, there is an effort underway to make species identification more readily available across a broad range of taxa through the sequencing of a standard gene. This effort requires the development of new methodologies to both rapidly produce and analyse large numbers of sequences. The methodology presented in this paper addresses these issues with a focus on the larvae of large pelagic fish species. All steps of the methodology are targeted towards high‐throughput identification using small amounts of tissue. To accomplish this, DNA isolation was automated on a liquid‐handling robot using magnetic bead technologies. Polymerase chain reaction and a unidirectional sequencing reaction followed standard protocols with all template cleanup and transferring also automated. Manual pipetting was thus reduced to a minimum. A character‐based bioinformatics program was developed to handle the large sequence output. This program incorporates base‐call quality scores in two types of sample to voucher sequence comparisons and provides suggested identifications and sequence information in an easily interpreted spreadsheet format. This technique when applied to tuna and billfish larvae collected in the Straits of Florida had an 89% success rate. A single species (Thunnus atlanticus) was found to dominate the catch of tuna larvae, while billfish larvae were more evenly divided between two species (Makaira nigricans and Istiophorus platypterus).     

High‐throughput mAb expression and purification platform based on transient CHO

A high‐cell‐density transient transfection system was recently developed in our laboratory based on a CHO‐GS‐KO cell line. This method yields monoclonal antibody titers up to 350 mg/L from a simple 7‐day process, in volumes ranging from 2 mL to 2 L. By performing transfections in 24‐deep‐well plates, a large number of mAbs can be expressed simultaneously. We coupled this new high‐throughput transfection process to a semiautomated protein A purification process. Using a Biomek FX^p liquid handling robot, up to 72 unique mAbs can be simultaneously purified. Our primary goal was to obtain >0.25 mg of purified mAb at a concentration of >0.5 mg/mL, without any concentration or buffer‐exchange steps. We optimized both the batch‐binding and the batch elution steps. The length of the batch‐binding step was important to minimize mAb losses in the flowthrough fraction. The elution step proved to be challenging to simultaneously maximize protein recovery and protein concentration. We designed a variable volume elution strategy based on the average supernatant titer. Finally, we present two case studies. In the first study, we produced 56 affinity maturation mAb variants at an average yield of 0.33 ± 0.05 mg (average concentration of 0.65 ± 0.10 mg/mL). In a second study, we produced 42 unique mAbs, from an early‐stage discovery effort, at an average yield of 0.79 ± 0.31 mg (average concentration of 1.59 ± 0.63 mg/mL). The combination of parallel high‐yielding transient transfection and semiautomated high‐throughput protein A purification represents a valuable mAb drug discovery tool. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:239–247, 2015     

High‐throughput CRISPRi phenotyping identifies new essential genes in Streptococcus pneumoniae

Genome‐wide screens have discovered a large set of essential genes in the opportunistic human pathogen Streptococcus pneumoniae. However, the functions of many essential genes are still unknown, hampering vaccine development and drug discovery. Based on results from transposon sequencing (Tn‐seq), we refined the list of essential genes in S. pneumoniae serotype 2 strain D39. Next, we created a knockdown library targeting 348 potentially essential genes by CRISPR interference (CRISPRi) and show a growth phenotype for 254 of them (73%). Using high‐content microscopy screening, we searched for essential genes of unknown function with clear phenotypes in cell morphology upon CRISPRi‐based depletion. We show that SPD_1416 and SPD_1417 (renamed to MurT and GatD, respectively) are essential for peptidoglycan synthesis, and that SPD_1198 and SPD_1197 (renamed to TarP and TarQ, respectively) are responsible for the polymerization of teichoic acid (TA) precursors. This knowledge enabled us to reconstruct the unique pneumococcal TA biosynthetic pathway. CRISPRi was also employed to unravel the role of the essential Clp‐proteolytic system in regulation of competence development, and we show that ClpX is the essential ATPase responsible for ClpP‐dependent repression of competence. The CRISPRi library provides a valuable tool for characterization of pneumococcal genes and pathways and revealed several promising antibiotic targets.     

High‐throughput sequencing of Hop stunt viroid‐derived small RNAs from cucumber leaves and phloem

Small RNA (sRNA)‐guided processes, referred to as RNA silencing, regulate endogenous and exogenous gene expression. In plants and some animals, these processes are noncell autonomous and can operate beyond the site of initiation. Viroids, the smallest self‐replicating plant pathogens known, are inducers, targets and evaders of this regulatory mechanism and, consequently, the presence of viroid‐derived sRNAs (vd‐sRNAs) is usually associated with viroid infection. However, the pathways involved in the biogenesis of vd‐sRNAs are largely unknown. Here, we analyse, by high‐throughput pyrosequencing, the profiling of the Hop stunt viroid (HSVd) vd‐sRNAs recovered from the leaves and phloem of infected cucumber (Cucumis sativus) plants. HSVd vd‐sRNAs are mostly 21 and 22 nucleotides in length and derived equally from plus and minus HSVd RNA strands. The widespread distribution of vd‐sRNAs across the genome reveals that the totality of the HSVd RNA genome contributes to the formation of vd‐sRNAs. Our sequence data suggest that viroid‐derived double‐stranded RNA functions as one of the main precursors of vd‐sRNAs. Remarkably, phloem vd‐sRNAs accumulated preferentially as 22‐nucleotide species with a consensus sequence over‐represented. This bias in size and sequence in the HSVd vd‐sRNA population recovered from phloem exudate suggests the existence of a selective trafficking of vd‐sRNAs to the phloem tissue of infected cucumber plants.     

High‐throughput genotyping of single‐nucleotide polymorphisms in ace‐1 gene of mosquitoes using MALDI‐TOF mass spectrometry

Abstract Acetylcholinesterase (AChE) plays a vital role in the nervous system of insects and other animal species and serves as the target for many chemical agents such as organophosphate and carbamate insecticides. The mosquito, Culex pipiens complex, a vector of human disease, has evolved to be resistant to insecticides by a limited number of amino acid substitutions in AChE1, which is encoded by the ace‐1 gene. The aims of this study are to identify single nucleotide polymorphism (SNP) sites in the ace‐1 gene of the C. pipiens complex and explore an economical high‐throughput method to differentiate the genotypes of these sites in mosquitoes collected in the field. We identified 22 SNP sites in exon regions of the ace‐1 gene. Four of them led to non‐synonymous mutations, that is, Y163C, G247S, C677S and T682A. We used matrix‐assisted laser desorption ionization – time‐of‐flight mass spectrometry for genotyping at these four sites and another site F416V, which was relevant to insecticide resistance, in 150 mosquitoes collected from 15 field populations. We were able to synchronize analysis of the five SNP sites in each well of a 384‐well plate for each individual mosquito, thus decreasing the cost to one‐fifth of the routine analysis. Heterozygous genotypes at Y163C and G247S sites were observed in one mosquito. The possible influence of the five SNP sites on the activity or function of the enzyme is discussed based on the predicted tertiary structure of the enzyme.     

High‐throughput methods for visualizing the teleost skeleton: capturing autofluorescence of alizarin red

For nearly a century, Alizarin red S (alizarin sodium sulfonate) has been used by morphologists to stain calcified bone matrix. More recently, its traditional use has frequently been replaced by more modern techniques; however, its auto fluorescent property continues to contribute to research well beyond the context of bone development and regeneration. The purpose of this study is to describe detailed methods that can be used to capture the autofluorescence of Alizarin red‐stained mineralized tissues in juvenile zebrafish. These methods allow for in situ analyses of minute skeletal elements, such as pharyngeal teeth, and preclude the need for dissection.     

High‐throughput screening techniques for rapid PEG‐based precipitation of IgG4 mAb from clarified cell culture supernatant

Locating optimal protein precipitation conditions for complex biological feed materials is problematic. This article describes the application of a series of high‐throughput platforms for the rapid identification and selection of conditions for the precipitation of an IgG₄ monoclonal antibody (mAb) from a complex feedstock using only microliter quantities of material. The approach uses 96‐microwell filter plates combined with high‐throughput analytical methods and a method for well volume determination for product quantification. The low material, time and resource requirements facilitated the use of a full factorial Design of Experiments (DoE) for the rapid investigation into how critical parameters impact the IgG₄ precipitation. To aid the DoE, a set of preliminary range‐finding studies were conducted first. Data collected through this approach describing Polyethylene Glycol (PEG) precipitation of the IgG₄ as a function of mAb concentration, precipitant concentration, and pH are presented. Response surface diagrams were used to explore interactions between parameters and to inform selection of the most favorable conditions for maximum yield and purification. PEG concentrations required for maximum yield and purity were dependant on the IgG₄ concentration; however, concentrations of 14 to 20% w/v, pH 6.5, gave optimal levels of yield and purity. Application of the high‐throughput approach enabled 1,155 conditions to be examined with less than 1 g of material. The level of insights gained over such a short time frame is indicative of the power of microwell experimentation in allowing the rapid identification of appropriate processing conditions for key bioprocess operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010