High-throughput Activity Assay Research Articles

Characterization and engineering of the type 3 secretion system needle monomer from Salmonella through the construction and screening of a comprehensive mutagenesis library.

Protein production strategies in bacteria are often limited due to the need for cell lysis and complicated purification schemes. To avoid these challenges, researchers have developed bacterial strains capable of secreting heterologous protein products outside the cell, but secretion titers often remain too low for commercial applicability. Improved understanding of the link between secretion system structure and its secretory abilities can help overcome the barrier to engineering higher secretion titers. Here, we investigated this link with the PrgI protein, the monomer of the secretory channel of the type 3 secretion system (T3SS) of Salmonella enterica. Despite detailed knowledge of the PrgI needle's assembly and structure, little is known about how its structure influences its secretory capabilities. To study this, we recently constructed a comprehensive codon mutagenesis library of the PrgI protein utilizing a novel one-pot recombineering approach. We then screened this library for functional T3SS assembly and secretion titer by measuring the secretion of alkaline phosphatase using a high-throughput activity assay. This allowed us to construct a first-of-its-kind secretion fitness landscape to characterize the PrgI needle's mutability at each position as well as the mutations which lead to enhanced T3SS secretion. We discovered new design rules for building a functional T3SS as well as identified hypersecreting mutants. This work can be used to increase understanding of the T3SS's assembly and identify further targets for engineering. This work also provides a blueprint for future efforts to engineer other complex protein assemblies through the construction of fitness landscapes.IMPORTANCEProtein secretion offers a simplified alternative method for protein purification from bacterial hosts. However, the current state-of-the-art methods for protein secretion in bacteria are still hindered by low yields relative to traditional protein purification strategies. Engineers are now seeking strategies to enhance protein secretion titers from bacterial hosts, often through genetic manipulations. In this study, we demonstrate that protein engineering strategies focused on altering the secretion apparatus can be a fruitful avenue toward this goal. Specifically, this study focuses on how changes to the PrgI needle protein from the type 3 secretion system from Salmonella enterica can impact secretion titer. We demonstrate that this complex is amenable to comprehensive mutagenesis studies and that this can yield both PrgI variants with increased secretory capabilities and insight into the normal functioning of the type 3 secretion system.

Cell-Based Assay Approaches for Glycosaminoglycan Synthase High-Throughput Screening: Development and Applications.

Glycosaminoglycan synthases have immense potential in applications involving synthesis of oligosaccharides, using enzymatic approaches and construction of cell factories that produce polysaccharides as critical metabolic components. However, the use of high-throughput activity assays to screen for the evolution of these enzymes can be challenging because there are no significant changes in fluorescence or absorbance associated with glycosidic bond formation. Here, using incorporation of azido-labeled N-acetylhexosamine analogs into bacterial capsule polysaccharides via bacterial metabolism and bioorthogonal chemistry, fluorophores were specifically introduced onto cell surfaces. Furthermore, correlations between detectable fluorescence signals and the polysaccharide-synthesizing capacity of individual bacteria were established. Among 10 candidate genes, 6 members of the chondroitin synthase family were quickly identified in a recombinant Bacillus subtilis host strain. Additionally, directed evolution of heparosan synthase was successfully performed using fluorescence-activated cell sorting of recombinant Escherichia coli O10:K5(L):H4, yielding several mutants with increased activity. Cell-based approaches that selectively detect the presence or absence of synthases within an individual colony of bacterial cells, as well as their level of activity, have broad potential in the exploration and engineering of glycosaminoglycan synthases. These approaches also support the creation of novel strategies for high-throughput screening of enzyme activity based on cell systems.

Expression element optimization and molecular modification to enhance the secretory expression of chitinase from Bacillus licheniformis in Bacillus subtilis

Chitin is the second most abundant polysaccharide in nature after cellulose. The degradation products of chitin, amino-oligosaccharides, possess good biological activities and promising applications. Chitinase plays an important role in the degradation of chitin, and its highly efficient production has attracted wide attention. In this study, the Bacillus licheniformis-derived chitinase BlChiA was recombinantly expressed in Bacillus subtilis. The optimal promoter SpoVG and signal peptide YqxI were obtained by screening the endogenous promoters and signal peptides. The extracellular enzyme activity of the recombinant strain reached 1.96 U mL−1, 1.94-fold higher than that of the original, previously constructed strain. On this basis, mutant libraries were constructed using error-prone PCR, and mutants with enhanced activities were obtained using high-throughput activity assays. After combination, the mutant E336K/A472T exhibited 3.61-fold greater activity than the previous result, which represented the highest expression level of BlChiA in Bacillus subtilis. In addition, the mutant also exhibited greater acid adaptability. These results provided a foundation for further exploration of the function and industrial application of the enzyme.

Improved Activity of Sulfolobus acidocaldarius Maltooligosyltrehalose Synthase through Directed Evolution.

Maltooligosyltrehalose synthase (MTSase) is a key enzyme for the production of trehalose from starch. Thermophilic MTSases offer advantages for trehalose production but suffer from low yield. In this study, directed evolution was used to increase the production of Sulfolobus acidocaldarius MTSase (SaMTSase) in Escherichia coli. Mutant libraries constructed using error-prone polymerase chain reaction were assessed using high-throughput activity assays. Three mutants with enhanced activities were obtained, the best of which (mutant D-4) exhibited 2.4 times greater activity than wild-type SaMTSase. The specific activity and catalytic efficiency of D-4 were also greater than those of wild-type SaMTSase. The D-4 activity (624.7 U·mL-1) produced in a 3 L fermenter was 2.0 times greater than that of wild-type SaMTSase. Because the same trehalose yield was obtained using an equal amount of either D-4 or wild-type SaMTSase activity, using D-4 will significantly lower the cost of trehalose production. The activities of the individual mutations present in the three SaMTSase mutants obtained using directed evolution were analyzed. Mutants F284V and T439A exhibited the greatest increases in enzyme activity. Homology models suggested that the decreased side-chain size, weakened hydrophobicity, and decreased interaction might enhance the flexibility of the loop containing catalytic residue Asp443, which was conducive to catalysis.

Enhancing the activity and thermostability of Streptomyces mobaraensis transglutaminase by directed evolution and molecular dynamics simulation

Streptomyces mobaraensis transglutaminase (MTG) has been extensively used in food industry and other biotechnological fields due to its cross-linking modification of proteins. Thus a MTG variant with a higher thermostability as well as an enhanced activity is desired because some processing for MTG applications is often performed at higher temperatures. In this study, the activity and thermostability of wild-type MTG (WT) were enhanced via directed evolution using error-prone PCR, and the mutant MTG (E164L) with the improved specific activity and thermostability was obtained using a high-throughput activity assay. E164L exhibited a 1.95 times specific activity of WT at 50 °C. Meanwhile, the half-life of E164L at 50 °C was 1.66-fold of WT. The molecular dynamics (MD) simulation results indicated that the mutation Glu164Leu resulted in the weaker interactions of Asp159-Glu164 and Gly228-Leu231, leading to the enhanced instability of Ile240-Asn253 linked to Gly228-Leu231 by eight residues. It further caused the reduced interactions between loop region 1 (Ile240-Asn253) and loop region 2 (His277-Met288), facilitating the access of the substrate molecule to the active site. This study improved the understanding of structure-activity relationship for MTG adapted to high temperature conditions. It also provides theoretical foundation and preliminary information for engineering MTG with enhanced characteristics to meet the industrial requirements.

Directed evolution of α-amylase from Bacillus licheniformis to enhance its acid-stable performance

α-Amylases (1,4-α-D-glucanohydrolases) are widely used in starch liquefaction, but their acid stability needs to be continuously explored to reduce the costs of raw material and operation. In this study, to better meet the industrial requirements, the acid stability of Bacillus licheniformis α-amylase (BLA) was further improved by directed evolution using error prone polymerase chain reaction (PCR). The mutant BLA (G81R) was selected with the improved acid stability based on a high-throughput activity assay. After incubating at pH 4.5 for 40 min, G81R still retained 10% of its initial activity, but the wild-type (WT) was already inactive. The kcat/Km value of G81R at pH 4.5 was 1.4-fold higher than that of WT. Combined with the three-dimensional structural modeling analysis, the improved stability of G81R under low pH condition might be due to the interactions of electrostatic, hydrophilicity, and helix propensity. Therefore, these findings would be beneficial for developing BLA with properties suitable for applications in industrial starch processing.

Improvement of the alkali stability of Penicillium cyclopium lipase by error-prone PCR

Background: Lipases are extensively exploited in lots of industrial fields; cold-adapted lipases with alkali-resistance are especially desired in detergent industry. Penicillium cyclopium lipase I (PCL) might be suitable for applications of detergent industry due to its high catalytic efficiency at low temperature and relatively good alkali stability. In this study, to better meet the requirements, the alkali stability of PCL was further improved via directed evolution with error-prone PCR. Results: The mutant PCL (N157F) with an improved alkali stability was selected based on a high-throughput activity assay. After incubating at pH 11.0 for 120 min, N157F retained 70% of its initial activity, which was 23% higher than that of wild type PCL. Combined with the three-dimensional structure analysis, N157F exhibited an improved alkali stability under the high pH condition due to the interactions of hydrophilicity and β-strand propensity. Conclusions: This work provided the theoretical foundation and preliminary data for improving alkali stability of PCL to meet the industrial requirements, which is also beneficial to improving alkali-tolerance ability of other industrial enzymes via molecular modification.

Improvement of the alkali stability of Penicillium cyclopium lipase by error-prone PCR

Background: Lipases are extensively exploited in lots of industrial fields; cold-adapted lipases with alkali-resistance are especially desired in detergent industry. Penicillium cyclopium lipase I (PCL) might be suitable for applications of detergent industry due to its high catalytic efficiency at low temperature and relatively good alkali stability. In this study, to better meet the requirements, the alkali stability of PCL was further improved via directed evolution with error-prone PCR. Results: The mutant PCL (N157F) with an improved alkali stability was selected based on a high-throughput activity assay. After incubating at pH 11.0 for 120 min, N157F retained 70% of its initial activity, which was 23% higher than that of wild type PCL. Combined with the three-dimensional structure analysis, N157F exhibited an improved alkali stability under the high pH condition due to the interactions of hydrophilicity and β-strand propensity. Conclusions: This work provided the theoretical foundation and preliminary data for improving alkali stability of PCL to meet the industrial requirements, which is also beneficial to improving alkali-tolerance ability of other industrial enzymes via molecular modification.

Human cytochrome P450 expression in bacteria: Whole-cell high-throughput activity assay for CYP1A2, 2A6 and 3A4

Cytochrome P450s (CYPs) are key enzymes involved in drug and xenobiotic metabolism. A wide array of in vitro methodologies, including recombinant sources, are currently been used to assess CYP catalysis, to identify the metabolic profile of compounds, potential drug-drug interactions, protein-protein interactions in the CYP enzyme complex and the role of polymorphic enzymes. We report here on a bacterial whole-cells high-throughput method for the activity evaluation of human CYP1A2, 2A6, and 3A4, when sustained by NADPH cytochrome P450 oxidoreductase (CPR), in the absence or presence of cytochrome b5 (CYB5). This new assay consists of a microplate real-time fluorometric method, with direct measurement of metabolite formation, in a suspension of Escherichia coli BTC-CYP bacteria, a human CYP competent tester strain when incubated with specific fluorogenic substrates. Overall, the maximum turnover (kcat) velocities of the three human CYPs resulting from the whole-BTC cells assays were similar to those obtained when applying the corresponding standard reference membrane fractions assays. CYP activity screening with co-expression of CYB5 suggests an enhancing effect of CYB5 on the kcat of specific isoforms, when using the whole-BTC cells assay. Our results demonstrate that this new approach can offer an efficient high-throughput method for screening of CYP1A2, 2A6 and 3A4 activity and can be potentially applicable for other human CYPs. This can be of particular use for timely and efficient screening of chemical libraries or mutant libraries of CYP enzyme complex proteins, without the necessity for labor intensive isolation of subcellular fractions.

Development of GC-MS based cytochrome P450 assay for the investigation of multi-herb interaction

As drug interactions with cytochrome P450 enzymes become increasingly important in the field of drug discovery, a high-throughput screening method for analysing the effects of a drug is needed. We have developed a simple and rapid simultaneous analytical method using a cocktail approach for measuring the activities of seven cytochrome P450 enzymes (CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and CYP3A4). Human liver microsomes were used as a source for the seven cytochrome P450 enzymes, and a gas chromatography-mass spectrometry (GC-MS) was used for analysing their activities. Kinetic studies and inhibition assays of CYP enzymes were performed using known substrates and inhibitors for validating and comparing the reaction rates and time-dependent activities between methods using each substrate versus a method using a cocktail solution. The optimized cocktail method was successfully applied to evaluate the effects of the decoction of Socheongryong-tang (SCRT) on cytochrome P450 enzymes. Our cocktail method provides a simultaneous high-throughput activity assay using GC-MS for the first time. This method is applicable for analysing the drug interactions of various plant-derived mixtures.

A novel high-throughput activity assay for the Trypanosoma brucei editosome enzyme REL1 and other RNA ligases.

The protist parasite Trypanosoma brucei causes Human African trypanosomiasis (HAT), which threatens millions of people in sub-Saharan Africa. Without treatment the infection is almost always lethal. Current drugs for HAT are difficult to administer and have severe side effects. Together with increasing drug resistance this results in urgent need for new treatments. T. brucei and other trypanosomatid pathogens require a distinct form of post-transcriptional mRNA modification for mitochondrial gene expression. A multi-protein complex called the editosome cleaves mitochondrial mRNA, inserts or deletes uridine nucleotides at specific positions and re-ligates the mRNA. RNA editing ligase 1 (REL1) is essential for the re-ligation step and has no close homolog in the mammalian host, making it a promising target for drug discovery. However, traditional assays for RELs use radioactive substrates coupled with gel analysis and are not suitable for high-throughput screening of compound libraries. Here we describe a fluorescence-based REL activity assay. This assay is compatible with a 384-well microplate format and sensitive, satisfies statistical criteria for high-throughput methods and is readily adaptable for other polynucleotide ligases. We validated the assay by determining kinetic properties of REL1 and by identifying REL1 inhibitors in a library of small, pharmacologically active compounds.

A plate-based high-throughput activity assay for polysialyltransferase from Neisseria meningitidis

Polysialyltransferases (PSTs) assemble polysialic acid (PSA) and have been implicated in many biological processes. For example, certain bacteria such as neuroinvasive Neisseria meningitidis decorate themselves in a PSA capsule to evade the innate immune system. Identifying inhibitors of PSTs therefore represents an attractive therapeutic goal and herein we describe a high-throughput, robust, and sensitive microtiter-plate-based activity assay for PST from N. meningitidis. A trisialyl lactoside (GT3) serving as the acceptor substrate was immobilized on a 384-well plate by click chemistry. Incubation with PST and CMP-sialic acid for 30min resulted in polysialylation. The immobilized PSA was then directly detected using a green fluorescent protein (GFP)-fused PSA-binding protein consisting of the catalytically inactive double mutant of an endosialidase (GFP-EndoNF DM). We report very good agreement between kinetic and inhibition parameters obtained with our on-plate assay versus our in-solution validation assay. In addition we prove our assay is robust and reliable with a Z′ score of 0.79. All aspects of our assay are easily scalable owing to optimization trials that allowed immobilization of acceptor substrates prepared from crude reaction mixtures and the use of cell lysates. This assay methodology enables large-scale PST inhibitor screens and can be harnessed for directed evolution screens.

Improved activity and thermostability of Bacillus pumilus lipase by directed evolution

To improve enzymatic activity of Bacillus pumilus lipases, DNA shuffling was applied to two lipase genes from local B. pumilus isolates. Using a high-throughput activity assay, the mutant with highest activity was selected. This chimeric mutant (L3-3), carrying two crossover positions and three point mutations, has a specific activity 6.4 and 8.2 times higher than the two parent enzymes. The mutant also is more tolerant to various detergents and organic solvents, and has a 9 times longer half-life at 50°C. Homology modeling of mutant L3-3, based on the highly homologous B. subtilis lipase A, shows that the increased thermostability is likely due to structural rigidification and reduced surface hydrophobicity. Increased specific activity may result from the location of mutations close to the active site. Together, our results show that it is possible to evolve, by DNA shuffling, B. pumilus lipase variants with improved applicability as biocatalysts, even if the two parent enzymes are highly similar.

O-GlcNAc Transferase and Its Inhibitors

O-GlcNAc transferase (OGT), one of essential mammalian enzymes, catalyses the transfer of N-acetylglucos- amine from UDP-N-acetylglucosamine (UDP-GlcNAc) to hydroxyl groups of serines and threonines (Ser/Thr) in proteins. O-GlcNAcylation is widely located in cytoplasm and nucleus. This kind of protein post-translational modification is involved in the regulation of many cellular signaling pathways and closely associated with the occurrences and developments of nu- merous critical illnesses. In animals, OGT is encoded by a single highly conserved gene. However, human OGT includes three different isoforms at least: nucleocytoplasmic OGT (ncOGT), mitochondrial OGT (mOGT) and short OGT (sOGT). All of the isoforms comprise two distinct regions: a multidomain catalytic region and an N-terminal region with different num- bers of tetratricopeptide repeats (TPRs), which is closely related to their subcellular localizations. The structures of the cata- lytic region and TPRs were reported and it was supported that OGT uses an ordered sequential bi-bi mechanism though the details of catalytic mechanism were not completely understood. To discover the inhibitors of OGT, several OGT activity assay methods were developed. The conventional method uses a radiolabeled glycosyl donor substrate such as UDP-( 3 H)-GlcNAc or UDP-( 14 C)-GlcNAc so that it is not suitable for in vivo test or rapid analysis. O-GlcNAc anti- body-based western blot was performed to measure the activity of OGT in vivo, but it is infeasible to determine low abun- dance protein with single O-GlcNAc. Three high throughput activity assays were reported: ligand displacement OGT assay, protease-protection assay strategy and azido-enzyme-linked immunosorbent assay (azido-ELISA). The first two methods have especially been used to find new OGT inhibitors through screening compound libraries, but the results have to be veri- fied by the conventional method. To date, OGT inhibitor activity of several candidates was evaluated and as found, two of them {2-acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-5-thio-α-D-glucopyranose and 4-methoxyphenyl 6-acetyl-2-oxobenzo(d)- oxazole-3(2H)-carboxylate} worked effectively in vivo and were valuable for understanding the functional roles of OGT. Nevertheless, there are still important questions to be answered in the research of OGT: (1) What is the general base that catalyzes O-GlcNAcylation reaction? (2) How can OGT recognize so many protein substances with the preservation of sub- stance specificity? (3) How can we develop robust high-throughput OGT assays and isoform-specific OGT inhibitors? In conclusion, there is a long way to go for comprehensive understanding of OGT.

PC6 levels in uterine lavage are closely associated with uterine receptivity and significantly lower in a subgroup of women with unexplained infertility

BACKGROUND Embryo implantation requires a healthy embryo and a receptive uterus. Uterine incompetence contributes significantly to implantation failure and infertility. To date, there are no reliable biochemical methods that can determine whether the uterus is receptive. Proprotein convertase 5/6 (PC6) is tightly regulated in the uterus and critical for receptivity and implantation; its secretory nature predicts PC6 to be secreted into the uterine cavity. The present study examines whether PC6 is detectable in uterine lavage and whether there is any correlation between secreted PC6 levels and uterine receptivity. METHODS Western blotting determined the presence of PC6 protein in uterine lavage. A sensitive and high-throughput activity assay was established and validated. This assay was applied to 103 lavages collected from different phases of the menstrual cycle from women with proven fertility or unexplained infertility. RESULTS Uterine lavage contained PC6 protein with levels paralleling enzymatic activity. PC6 levels were significantly higher in the receptive than in the non-receptive phase in fertile women, and the putative receptive phase levels in a subgroup of women with unexplained infertility were significantly lower than in the fertile counterparts. CONCLUSIONS PC6 levels in uterine lavage are significantly elevated in the luteal phase of fertile women and markedly reduced in a subgroup of women with unexplained infertility. Uterine fluid is a valuable source of material to evaluate uterine function. Detection of PC6 in uterine fluid may lead to the development of a rapid and relatively non-surgical assessment of uterine receptivity.

The activity of wild-type and mutant phenylalanine hydroxylase and its regulation by phenylalanine and tetrahydrobiopterin at physiological and pathological concentrations: An isothermal titration calorimetry study

The activity of phenylalanine hydroxylase (PAH) is regulated by the levels of both the substrate ( l-Phe) and the natural cofactor (6 R)-tetrahydrobiopterin (BH 4). It has recently been observed that many PAH mutants associated with BH 4-responsive phenylketonuria display abnormal kinetic and regulatory properties as shown by standard kinetic analyses. In this work, we have developed a high-sensitive and high-throughput activity assay based on isothermal titration calorimetry (ITC) in order to study the kinetic properties of wild-type PAH (wt-PAH) and the BH 4-responsive c.204A > T (p.R68S) mutant at physiological and superphysiological concentrations of l-Phe and BH 4. Compared to wt-PAH, the p.R68S mutant showed reduced apparent and equilibrium binding affinity for the natural cofactor and increased affinity and non-cooperative response for l-Phe, together with a strong substrate inhibition that is alleviated at high BH 4 concentrations. For both wt-PAH and mutant, the apparent affinity for BH 4 decreases at increasing l-Phe concentrations, and the affinity for the substrate also depends on the cofactor concentration. Our results indicate that the activity landscape for wt and mutant enzymes is more complex than expected from standard kinetic analyses and highlight the applicability of this ITC-based assay to characterize the activity and regulation of PAH at a wide range of substrate and cofactor concentrations. Moreover, the results aid to understand the activity dynamics of wild-type and mutant PAH under physiological and pathological conditions, as well as BH 4-responsiveness in certain PKU mutations.

A Novel, High Performance Enzyme for Starch Liquefaction: DISCOVERY AND OPTIMIZATION OF A LOW pH, THERMOSTABLE α-AMYLASE

High throughput screening of microbial DNA libraries was used to identify alpha-amylases with phenotypic characteristics compatible with large scale corn wet milling process conditions. Single and multiorganism DNA libraries originating from various environments were targeted for activity and sequence-based screening approaches. After initial screening, 15 clones were designated as primary hits based upon activity at pH 4.5 or 95 degrees C without addition of endogenous Ca(2+). After further characterization, three enzyme candidates were chosen each with an exceptional expression of one or more aspects of the necessary phenotype: temperature stability, pH optimum, lowered reliance on Ca(2+) and/or enzyme rate. To combine the best aspects of the three phenotypes to optimize process compatibility, the natural gene homologues were used as a parental sequence set for gene reassembly. Approximately 21,000 chimeric daughter sequences were generated and subsets screened using a process-specific, high throughput activity assay. Gene reassembly resulted in numerous improved mutants with combined optimal phenotypes of expression, temperature stability, and pH optimum. After biochemical and process-specific characterization of these gene products, one alpha-amylase with exceptional process compatibility and economics was identified. This paper describes the synergistic approach of combining environmental discovery and laboratory evolution for identification and optimization of industrially important biocatalysts.