Random Library Research Articles

High-Throughput Discovery of Substrate Peptide Sequences for E3 Ubiquitin Ligases Using a cDNA Display Method.

Cells utilize ubiquitin as a posttranslational protein modifier to convey various signals such as proteasomal degradation. The disfunction of ubiquitylation or following proteasomal degradation can give rise to the accumulation and aggregation of improperly ubquitylated proteins, which is known to be a general causation of many neurodegenerative diseases. Thus, the characterization of substrate peptide sequences of E3 ligases is crucial in biological and pharmaceutical sciences. In this study, we developed a novel high-throughput screening system for substrate peptide sequences of E3 ligases using a cDNA display method, which enables covalent conjugation between peptide sequences and their corresponding cDNA sequences. First, we focused on the MDM2 E3 ligase and its known peptide substrate as a model to establish the screening method, and confirmed that cDNA display method was compatible with in vitro ubiquitylation. Then, we demonstrated identification of MDM2 substrate sequences from random libraries to identify a novel motif (VKFTGGQLA). Bioinformatics analysis of the hit sequences was performed to gain insight about endogenous substrate proteins.

A yeast surface display platform for screening of non-enzymatic protein secretion in Kluyveromyces lactis.

Enhancing the secretion of recombinant proteins, particularly non-enzymatic proteins that predominate in food and pharmaceutic protein products, remains a significant challenge due to limitations in high-throughput screening methods. This study addresses this bottleneck by establishing a yeast surface display system in the food-grade microorganism Kluyveromyces lactis, enabling efficient display of model target proteins on the yeast cell surface. To assess its potential as a universal high-throughput screening tool for enhanced non-enzymatic protein secretion, we evaluated the consistency between protein display levels and secretion efficiency under the influence of various genetic factors. Our results revealed a strong correlation between these two properties. Furthermore, screening in a random mutagenesis library successfully identified a mutant with improved secretion. These findings demonstrate the potential of the K. lactis surface display system as a powerful and universal tool for high-throughput screening of strains with superior non-enzymatic protein secretion capacity. We believe this study could pave the way for efficient large-scale production of heterologous food and therapeutic proteins in industries. KEY POINTS: • A YSD (yeast surface display) system was established in Kluyveromyces lactis • This system enables high-throughput screening of non-enzymatic protein secretion • This technology assists industrial production of food and therapeutic proteins.

Molecular Engineering L-Aspartate-Alpha-Decarboxylase to Enhance Catalytic Stability and Performance.

L-aspartate-alpha-decarboxylase (ADC) catalyzes the decarboxylation of L-aspartate to produce β-alanine, which is the decisive step in the biosynthesis of β-alanine. However, the low catalytic stability and efficiency of ADC limit its industrial applications. In this study, a variant of ADC from Bacillus subtilis were used as a starting point for engineering. After constructing a random mutagenesis library by error-prone PCR, followed by high-throughput screening,four substitutions (S7 N, K63 N, A99T, and K113R) were identified. By screening saturation mutagenesis libraries on these positions and computational analysis, two recombined variants N3(S7 N/K63 N/I88 M/A99E/K113R/I126*) and Y1(S7Y/K63 N/I88 M/A99E/K113R/I126*) with improved performance were obtained. Compared to the wild type, the catalytic efficiency and catalytic stability of the best two variants were enhanced up to 95 %(variant N3) and up to 89 %(variant Y1), respectively. In addition, Y1 exhibited 3.37 times improved half-life and 2-fold improved total turnover number. Hydrophilicity analysis and molecular dynamics (MD) simulation revealed that the increased hydrophilicity and steric hindrance of key amino acid residues would affect the catalytic activity and stability. The improved catalytic performance of the variants could be attributed to their enhanced binding capacity to the substrate within the active pocket and the alleviation of mechanism-based inactivation.

Electrochemical aptasensor for 2-amino-2-thiazoline-4-carboxylic acid (ATCA), a metabolite for cyanide poisoning

An alternative biomarker for assessing the cyanide levels in postmortem materials is crucial for investigating acute cyanide intoxication. Herein, an aptamer–ligand biorecognition system with high specificity was developed to detect acute cyanide poisoning via its secondary metabolite, 2-amino-2-thiazoline-4-carboxylic acid (ATCA). Potential aptamers were screened from a random library of 66-base single-stranded DNA using GO-SELEX, with individual aptamers being identified through single-stranded DNA sequencing. Molecular docking was employed to predict the affinity of these aptamers toward ATCA and selected counter-targets; these predictions were confirmed using thermodynamic analysis with an isothermal titration calorimeter. Owing to its label-free biomolecular binding interactions, Apt46 exhibited the highest affinity against ATCA and notable selectivity against structurally similar counter-targets. Thus, an amino-tagged Apt46 binding aptamer was attached to a carbon electrode modified with EDC–NHS-activated graphene oxide. The binding of Apt46 to ATCA was quantified by measuring current changes using differential pulse voltammetry. The aptasensor achieved a detection limit of 0.05 µg/mL and demonstrated suitability for detecting ATCA across various biological matrices, with the high recovery percentages ranging from 92.29 to 114.22%. Overall, the proposed ATCA aptasensor is promising for identifying ATCA metabolites in cases of acute cyanide exposure.

Selection of antibacterial aptamers against Pseudomonas plecoglossicida and analysis on their potential binding proteins

Pseudomonas plecoglossicida is one of the main pathogens causing visceral white spot disease of the large yellow croaker (Pseudosciaena crocea). Although antibiotics are used to control P. plecoglossicida infections, the long-term and large-scale use of antibiotics can lead to the development of drug-resistant bacteria as well as environmental pollution. Therefore, it is necessary to develop novel antibacterial agents to treat P. plecoglossicida infections. In this study, we first developed a two-step-centrifugation method to isolate live and dead P. plecoglossicida cells. Subsequently, we used the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) screening based on inhibition rate to directly isolate antibacterial aptamers from the random library without post-processing. We isolated five antibacterial aptamers, namely B1, B2, B4, B8, and B109, which showed good inhibitory effects against P. plecoglossicida. Among these, B4 showed the highest inhibitory effect (62.40 ± 4.17 %). Further analysis revealed no positive correlation between the inhibitory effect of aptamers and their affinities with the target bacterium. The B4- and B109-binding proteins were isolated from P. plecoglossicida by magnetic separation and sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). Mass spectrometry analysis revealed that the 26 kDa ribosomal protein L2 was the probable B4-binding protein, while the 26 kDa ribosomal protein S3 and 75 kDa ribosomal protein S1 or succinate dehydrogenase flavoprotein subunit were the probable B109-binding proteins. Structural and subcellular localization analyses of these potential B4- and B109-binding proteins were also conducted. Our findings suggest that the inhibitory activity of the antibacterial aptamers against P. plecoglossicida may be mediated via their interaction with the ribosomal proteins, which can interfere with the protein synthesis process in the bacterium, affecting its growth. These findings provide the scientific basis for the development of functional antibacterial aptamers and the elucidation of their mechanisms of action.

Breeding of L-tryptophan high-yield strain by ARTP mutagenesis and high-throughput screening

L-tryptophan is an indispensable essential amino acid with a wide range of applications, which leads to a high demand. Accordingly, the production of L-tryptophan becomes a much-anticipated direction in research and industrial development. While irrational mutagenesis is an effective means to breed industrial strains, how to screen the strains with desirable phenotypes is still a major challenge. In order to improve the efficiency and accuracy of screening L-tryptophan high-yield strains, we used atmospheric and room temperature plasma mutagenesis to construct a random mutant library and then combined it with high-throughput screening in deep-well plates. Using a pseudo-fluorescent protein sensor capable of responding specifically to L-tryptophan, we successfully screened out a strain producing L-tryptophan at a high yield from a random mutagenesis library. The fermentation with the strain in shake flasks produced L-tryptophan at a yield of 1.99 g/L, which was 41.77% higher than that of the starting strain. Finally, the mechanism of high yield of the strain was deciphered by comparative genomics and transcriptomics. The above strategies provide a solid research foundation for further selection and development of high quality L-tryptophan producing strains.

High Throughput Screening of Transcription Factor LysG for Constructing a Better Lysine Biosensor.

The biosensors based on transcription factors (TFs) are widely used in high throughput screening of metabolic overproducers. The unsatisfactory performances (narrow detection and dynamic ranges) of biosensors limit their practical application and need more improvement. In this study, using the TF LysG (sensing lysine) as an example, a biosensor optimization method was constructed by growth-coupled screening of TF random mutant libraries. The better the performance of the biosensor, the faster the strain grows under screening pressure. A LysGE15D, A54D, and I164V-based biosensors were obtained, which were about 2-fold of the control in the detection and dynamic ranges. A lysine high-producer was screened effectively using the optimized biosensor with the production at 1.51 ± 0.30 g/L in flasks (2.22-fold of the original strain). This study provided a promising strategy for optimizing TF-based biosensors and was of high potential to be applied in the lysine high-producers screening process.

A yeast two-hybrid system to obtain triple-helical ligands from combinatorial random peptide libraries

Many bioactive proteins interact with collagen, recognizing amino acid sequences displayed on the triple helix. We report here a selection strategy to obtain triple-helical peptides that interact with the proteins from a combinatorial random library constructed in yeast cells. This system enables us to select them using the standard two-hybrid protocol, detecting interactions between triple-helical peptides and target proteins fused to the GAL4-activating and binding domains, respectively. The library was constructed having triple-helical peptides with a “host-guest” design in which host helix-stabilizing regions flanked guest random sequences. Using this system, we selected peptides that bind to pigment epithelium-derived factor (PEDF), a collagen-binding protein that shows anti-angiogenic and neurotrophic activities, from the libraries. Two-step selections from the total random library and subsequently from the second focused library yielded new PEDF-binding sequences that exhibited a comparable affinity to or more potent than that of the native PEDF-binding sequence in collagen. The obtained sequences also contained a variant of the PEDF-binding motif that did not match the known motif identified from the native collagen sequences. This combinatorial library system allows the chemical space of triple-helical peptides to be screened more widely than that found in native collagen, thus increasing the expectation of obtaining more specific and high-affinity peptides.

The efficient screening of avermectin B1a aptamer and colorimetric detection of its regulation of Ru NPs peroxidase-like activity

Avermectin is a dual-purpose antibiotic used in agriculture and livestock. Its widespread and massive use leads to its accumulation in the biological chain, causing significant impact on human food safety. Therefore, there is a need to establish a rapid and effective method for the detection of avermectin. In this study, a combination of the GO-SELEX and CE-SELEX methods was employed to screen aptamers for avermectin B1a from a ssDNA random library. After 7 rounds of selection, three aptamers with high enrichment and low homogeneity were chosen for affinity analysis, and the aptamer Seq 1 with the highest affinity was obtained. Optimized and truncated Seq 1 yielded the final high-affinity and specific aptamer, Seq 1–1. Based on the inhibitory effect of Seq 1–1 aptamer on the peroxidase-like activity of ruthenium nanoparticles (Ru NPs), for the first time, we developed a simple and stable colorimetric aptasensor for the detection of residual avermectin B1a. The linear range of the colorimetric sensor is 6.25–100 nM, with a minimum detection limit of 5.86 nM. In addition, the constructed sensor was used to detect avermectin B1a in milk and tomatoes, with a recovery rate of 92.86 %–101.49 %. This indicates that the sensor has broad application prospects in the detection of avermectin samples in animal husbandry and agriculture.

Culture Wars: Empirically Determining the Best Approach for Plasmid Library Amplification.

DNA libraries are critical components of many biological assays. These libraries are often kept in plasmids that are amplified in E. coli to generate sufficient material for an experiment. Library uniformity is critical for ensuring that every element in the library is tested similarly and is thought to be influenced by the culture approach used during library amplification. We tested five commonly used culturing methods for their ability to uniformly amplify plasmid libraries: liquid, semisolid agar, cell spreader-spread plates with high or low colony density, and bead-spread plates. Each approach was evaluated with two library types: a random 80-mer library, representing high complexity and low coverage of similar sequence lengths, and a human TF ORF library, representing low complexity and high coverage of diverse sequence lengths. We found that no method was better than liquid culture, which produced relatively uniform libraries regardless of library type. However, when libraries were transformed with high coverage, the culturing method had minimal impact on uniformity or amplification bias. Plating libraries was the worst approach by almost every measure for both library types and, counterintuitively, produced the strongest biases against long sequence representation. Semisolid agar amplified most elements of the library uniformly but also included outliers with orders of magnitude higher abundance. For amplifying DNA libraries, liquid culture, the simplest method, appears to be best.

Combination of error-prone PCR (epPCR) and Circular Polymerase Extension Cloning (CPEC) for improving the coverage of random mutagenesis libraries

Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library’s breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.

Chemical space exploration with Molpher: Generating and assessing a glucocorticoid receptor ligand library.

Computational exploration of chemical space is crucial in modern cheminformatics research for accelerating the discovery of new biologically active compounds. In this study, we present a detailed analysis of the chemical library of potential glucocorticoid receptor (GR) ligands generated by the molecular generator, Molpher. To generate the targeted GR library and construct the classification models, structures from the ChEMBL database as well as from the internal IMG library, which was experimentally screened for biological activity in the primary luciferase reporter cell assay, were utilized. The composition of the targeted GR ligand library was compared with a reference library that randomly samples chemical space. A random forest model was used to determine the biological activity of ligands, incorporating its applicability domain using conformal prediction. It was demonstrated that the GR library is significantly enriched with GR ligands compared to the random library. Furthermore, a prospective analysis demonstrated that Molpher successfully designed compounds, which were subsequently experimentally confirmed to be active on the GR. A collection of 34 potential new GR ligands was also identified. Moreover, an important contribution of this study is the establishment of a comprehensive workflow for evaluating computationally generated ligands, particularly those with potential activity against targets that are challenging to dock.

Attribute-based linear homomorphic signature scheme based on key policy for mobile crowdsensing

Compared with traditional wireless sensor networks, mobile crowdsensing networks have advantages of low cost, easy maintenance, and high scalability, which will play a role in city-level data sensing scenarios in the future. So far, linear homomorphic signatures based on Public Key Instruction,identity, as well as certificateless, have been proposed in wireless sensor networks to resist the data contamination. However, these signature schemes cannot perform finer-grained signature verification, and these signature schemes do not realize the separation of users’ sensitive information from their data. To solve the above problems, we design an attribute-based linear homomorphic signature scheme for large-scale wireless network built with mobile smart devices. First, we give the definition of the attribute-based linear homomorphic signature scheme based on key policy (KP-ABLHS). Second, we construct KP-ABLHS by incorporating attribute-based signature and linear homomorphic coding signature scheme. Finally, we prove our protocol is secure in random oracle model (ROM) and use Python pairing-based cryptography library (pypbc) to implement the scheme. The experimental results show that our scheme is as efficient as Li et al.’s scheme and has the advantage of signing the set of attributes, while the efficiency of our scheme is significantly better than that of scheme Boneh et al.’s scheme.

From Protein to a Bioactive Peptide: Potent IL‐6 Peptide Antagonist Generated by a Novel Method

ABSTRACTHere, we describe a new technology, designed to accelerate peptide discovery by quick identification and optimization of the residues critical for protein–protein interactions or ligand binding. We called it PepFusion. It is based on ligation of short DNA sequences generated from known ligand‐binding regions. We tested it by selecting peptide antagonists of interleukin‐6 (IL‐6), a key mediator of inflammatory diseases such as rheumatoid arthritis (RA), Crohn's disease, and Castleman disease (CD). The PepFusion library demonstrated superiority over a random library by yielding a peptide with low micromolar affinity for IL‐6, whereas the random library failed. The affinity of the peptide from the PepFusion library was further enhanced by additional rounds of mutagenesis leading to peptide variants with low nanomolar IL‐6 affinity. In addition to generating high‐affinity peptides, our method opens the way to solve the problem of the false positive sequences, which are common with all display technologies.

Computational design of Periplasmic binding protein biosensors guided by molecular dynamics.

Periplasmic binding proteins (PBPs) are bacterial proteins commonly used as scaffolds for substrate-detecting biosensors. In these biosensors, effector proteins (for example fluorescent proteins) are inserted into a PBP such that the effector protein's output changes upon PBP-substate binding. The insertion site is often determined by comparison of PBP apo/holo crystal structures, but random insertion libraries have shown that this can miss the best sites. Here, we present a PBP biosensor design method based on residue contact analysis from molecular dynamics. This computational method identifies the best previously known insertion sites in the maltose binding PBP, and suggests further previously unknown sites. We experimentally characterise fluorescent protein insertions at these new sites, finding they too give functional biosensors. Furthermore, our method is sufficiently flexible to both suggest insertion sites compatible with a variety of effector proteins, and be applied to binding proteins beyond PBPs.

Screening Key Sites of Class 2 Integron Integrase that Impact Recombination Efficiency.

By capturing and expressing exogenous resistance gene cassettes through site-specific recombination, integrons play important roles in the horizontal transfer of antimicrobial resistant genes among bacteria. The characteristics of integron integrase make it to be a potential gene editing tool enzyme. In this study, a random mutation library using error-prone PCR was constructed, and amino acid residues mutants that impact on attI2 × attC or attC × attC recombination efficiency were screened and analyzed. Thirteen amino acid mutations were identified to be critical impacted on site-specific recombination of IntI2, including the predicted catalyzed site Y301. Nine of 13 mutated amino acid residues that have critically impacted on IntI2 activity were relative concentrated and near the predicted catalyzed site Y301 in the predicted three-dimensional structure indicated the importance of this area in maintain the activity of IntI2. No mutant with obviously increased recombination activity (more than four-fold as high as that of wild IntI2) was found in library screening, except P95S, R100K slightly increased (within two-fold) the excision activity of IntI2, and S243T slightly increased (within two-fold) both excision and integration activity of IntI2. These findings will provide clues for further specific modification of integron integrase to be a tool enzyme as well as establishing a new gene editing system and applied practically.

Redefining the bacteriophage mv4 site-specific recombination system and the sequence specificity of its attB and core-attP sites.

Through their involvement in the integration and excision of a large number of mobile genetic elements, such as phages and integrative and conjugative elements (ICEs), site-specific recombination systems based on heterobivalent tyrosine recombinases play a major role in genome dynamics and evolution. However, despite hundreds of these systems having been identified in genome databases, very few have been described in detail, with none from phages that infect Bacillota (formerly Firmicutes). In this study, we reanalyzed the recombination module of Lactobacillus delbrueckii subsp. bulgaricus phage mv4, previously considered atypical compared with classical systems. Our results reveal that mv4 integrase is a 369 aa protein with all the structural hallmarks of recombinases from the Tn916 family and that it cooperatively interacts with its recombination sites. Using randomized DNA libraries, NGS sequencing, and other molecular approaches, we show that the 21-bp core-attP and attB sites have structural similarities to classical systems only if considering the nucleotide degeneracy, with two 7-bp inverted regions corresponding to mv4Int core-binding sites surrounding a 7-bp strand-exchange region. We also examined the different compositional constraints in the core-binding regions, which define the sequence space of permissible recombination sites.

Genome-Scale Investigation of the Regulation of azoR Expression in Escherichia coli Using Computational Analysis and Transposon Mutagenesis

Bacterial azoreductases are enzymes that catalyze the reduction of ingested or industrial azo dyes. Although azoreductase genes have been well identified and characterized, the regulation of their expression has not been systematically investigated. To determine how different factors affect the expression of azoR, we extracted and analyzed transcriptional data from the Gene Expression Omnibus (GEO) resource, then confirmed computational predictions by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Results showed that azoR expression was lower with higher glucose concentration, agitation speed, and incubation temperature, but higher at higher culture densities. Co-expression and clustering analysis indicated ten genes with similar expression patterns to azoR: melA, tpx, yhbW, yciK, fdnG, fpr, nfsA, nfsB, rutF, and chrR (yieF). In parallel, constructing a random transposon library in E. coli K-12 and screening 4320 of its colonies for altered methyl red (MR)-decolorizing activity identified another set of seven genes potentially involved in azoR regulation. Among these genes, arsC, relA, plsY, and trmM were confirmed as potential azoR regulators based on the phenotypic decolorization activity of their transposon mutants, and the expression of arsC and relA was confirmed, by qRT-PCR, to significantly increase in E. coli K-12 in response to different MR concentrations. Finally, the significant decrease in azoR transcription upon transposon insertion in arsC and relA (as compared to its expression in wild-type E. coli) suggests their probable involvement in azoR regulation. In conclusion, combining in silico analysis and random transposon mutagenesis suggested a set of potential regulators of azoR in E. coli.

Identification of Critical Amino Acid Residues of a Two-Component Sensor Protein for Signal Sensing in Porphyromonas gingivalis Fimbriation via Random Mutant Library Construction.

Porphyromonas gingivalis (Pg) utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS-FimR two-component system, making the FimS sensory protein an attractive target for preventing Pg infection. However, the specific environmental signal received by FimS remains unknown. We constructed random Pg mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of fimS, and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the fimS-knockout Pg strain using the Escherichia coli-Pg conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased fimA expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of Pg, including the elucidation of structure-function relationships of proteins of interest.

Predicting inhibitor development using a random peptide phage-display library approach in the SIPPET cohort

AbstractInhibitor development is the most severe complication of hemophilia A (HA) care and is associated with increased morbidity and mortality. This study aimed to use a novel immunoglobulin G epitope mapping method to explore the factor VIII (FVIII)–specific epitope profile in the SIPPET cohort population and to develop an epitope mapping–based inhibitor prediction model. The population consisted of 122 previously untreated patients with severe HA who were followed up for 50 days of exposure to FVIII or 3 years, whichever occurred first. Sampling was performed before FVIII treatment and at the end of the follow-up. The outcome was inhibitor development. The FVIII epitope repertoire was assessed by means of a novel random peptide phage-display assay. A least absolute shrinkage and selection operator (LASSO) regression model and a random forest model were fitted on posttreatment sample data and validated in pretreatment sample data. The predictive performance of these models was assessed by the C-statistic and a calibration plot. We identified 27 775 peptides putatively directed against FVIII, which were used as input for the statistical models. The C-statistic of the LASSO and random forest models were good at 0.78 (95% confidence interval [CI], 0.69-0.86) and 0.80 (95% CI, 0.72-0.89). Model calibration of both models was moderately good. Two statistical models, developed on data from a novel random peptide phage display assay, were used to predict inhibitor development before exposure to exogenous FVIII. These models can be used to set up diagnostic tests that predict the risk of inhibitor development before starting treatment with FVIII.