scholarly journals Optimization of the experimental parameters of the ligase cycling reaction

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Niels Schlichting ◽  
Felix Reinhardt ◽  
Sven Jager ◽  
Michael Schmidt ◽  
Johannes Kabisch
Keyword(s):  
Secondary Structure ◽  
Thermal Denaturation ◽  
Screening System ◽  
Experimental Procedure ◽  
Melting Temperatures ◽  
Assembly Method ◽  
High Efficient ◽  
Experimental Parameters ◽  
Molecular Crosstalk ◽  
Validation Experiments

Abstract The ligase cycling reaction (LCR) is a scarless and efficient method to assemble plasmids from fragments of DNA. This assembly method is based on the hybridization of DNA fragments with complementary oligonucleotides, so-called bridging oligos (BOs), and an experimental procedure of thermal denaturation, annealing and ligation. In this study, we explore the effect of molecular crosstalk of BOs and various experimental parameters on the LCR by utilizing a fluorescence-based screening system. The results indicate an impact of the melting temperatures of BOs on the overall success of the LCR assembly. Secondary structure inhibitors, such as dimethyl sulfoxide and betaine, are shown to negatively impact the number of correctly assembled plasmids. Adjustments of the annealing, ligation and BO-melting temperature further improved the LCR. The optimized LCR was confirmed by validation experiments. Based on these findings, a step-by-step protocol is offered within this study to ensure a routine for high efficient LCR assemblies.

scholarly journals Optimization of the experimental parameters of the ligase cycling reaction

2019 ◽  
Author(s):  
Niels Schlichting ◽  
Felix Reinhardt ◽  
Sven Jager ◽  
Michael Schmidt ◽  
Johannes Kabisch
Keyword(s):  
Secondary Structure ◽  
Thermal Denaturation ◽  
Screening System ◽  
Experimental Procedure ◽  
Melting Temperatures ◽  
Assembly Method ◽  
High Efficient ◽  
Experimental Parameters ◽  
Molecular Crosstalk ◽  
Validation Experiments

ABSTRACTThe ligase cycling reaction (LCR) is a scarless and efficient method to assemble plasmids from fragments of DNA. This assembly method is based on the hybridization of DNA fragments with complementary oligonucleotides, so-called bridging oligos (BOs), and an experimental procedure of thermal denaturation, annealing and ligation. In this study, we explore the effect of molecular crosstalk of BOs and various experimental parameters on the LCR by utilizing a fluorescence-based screening system. The results indicate an impact of the melting temperatures of BOs on the overall success of the LCR assembly. Secondary structure inhibitors, such as DMSO and betaine, are shown to negatively impact the number of correctly assembled plasmids. Adjustments of the annealing, ligation and BO-melting temperature further improved the LCR. The optimized LCR was confirmed by validation experiments. Based on these findings, a step-by-step protocol is offered within this study to ensure a routine for high efficient LCR assemblies.

Effect of Temperature and pH on the Secondary Structure and Denaturation Process of Jumbo Squid Hepatopancreas Cathepsin D.

2019 ◽  
Vol 26 (7) ◽  
pp. 532-541 ◽  
Author(s):  
Cadena-Cadena Francisco ◽  
Cárdenas-López José Luis ◽  
Ezquerra-Brauer Josafat Marina ◽  
Cinco-Moroyoqui Francisco Javier ◽  
López-Zavala Alonso Alexis ◽  
...  
Keyword(s):  
Circular Dichroism ◽  
Secondary Structure ◽  
Cathepsin D ◽  
Thermal Denaturation ◽  
Protein Denaturation ◽  
Marine Organisms ◽  
Effect Of Temperature ◽  
Jumbo Squid ◽  
Α Helix ◽  
Circular Dichroïsm

Background: Cathepsin D is a lysosomal enzyme that is found in all organisms acting in protein turnover, in humans it is present in some types of carcinomas, and it has a high activity in Parkinson's disease and a low activity in Alzheimer disease. In marine organisms, most of the research has been limited to corroborate the presence of this enzyme. It is known that cathepsin D of some marine organisms has a low thermostability and that it has the ability to have activity at very acidic pH. Cathepsin D of the Jumbo squid (Dosidicus gigas) hepatopancreas was purified and partially characterized. The secondary structure of these enzymes is highly conserved so the role of temperature and pH in the secondary structure and in protein denaturation is of great importance in the study of enzymes. The secondary structure of cathepsin D from jumbo squid hepatopancreas was determined by means of circular dichroism spectroscopy. Objective: In this article, our purpose was to determine the secondary structure of the enzyme and how it is affected by subjecting it to different temperature and pH conditions. Methods: Circular dichroism technique was used to measure the modifications of the secondary structure of cathepsin D when subjected to different treatments. The methodology consisted in dissecting the hepatopancreas of squid and freeze drying it. Then a crude extract was prepared by mixing 1: 1 hepatopancreas with assay buffer, the purification was in two steps; the first step consisted of using an ultrafiltration membrane with a molecular cut of 50 kDa, and the second step, a pepstatin agarose resin was used to purification the enzyme. Once the enzyme was purified, the purity was corroborated with SDS PAGE electrophoresis, isoelectric point and zymogram. Circular dichroism is carried out by placing the sample with a concentration of 0.125 mg / mL in a 3 mL quartz cell. The results were obtained in mdeg (millidegrees) and transformed to mean ellipticity per residue, using 111 g/mol molecular weight/residue as average. Secondary-structure estimation from the far-UV CD spectra was calculated using K2D Dichroweb software. Results: It was found that α helix decreases at temperatures above 50 °C and above pH 4. Heating the enzyme above 70°C maintains a low percentage of α helix and increases β sheet. Far-UV CD measurements of cathepsin D showed irreversible thermal denaturation. The process was strongly dependent on the heating rate, accompanied by a process of oligomerization of the protein that appears when the sample is heated, and maintained a certain time at this temperature. An amount typically between 3 and 4% α helix of their secondary structure remains unchanged. It is consistent with an unfolding process kinetically controlled due to the presence of an irreversible reaction. The secondary structure depends on pH, and a pH above 4 causes α helix structures to be modified. Conclusion: In conclusion, cathepsin D from jumbo squid hepatopancreas showed retaining up to 4% α helix at 80°C. The thermal denaturation of cathepsin D at pH 3.5 is under kinetic control and follows an irreversible model.

scholarly journals The Phenanthridine-modified Tyrosine Dipeptide

2019 ◽  
Vol 92 (2) ◽  
pp. 249-258
Author(s):  
Antonija Erben ◽  
Josipa Matić ◽  
Nikola Basarić ◽  
Ivo Piantanida
Keyword(s):  
Unnatural Amino Acids ◽  
Thermal Denaturation ◽  
Specific Binding ◽  
Quinone Methide ◽  
Melting Temperatures ◽  
Double Helices ◽  
Fluorescence Titrations ◽  
Alkylate Dna ◽  
Ct Dna ◽  
Phenanthridine Derivative

Dipeptide 4 containing two unnatural amino acids, a modified tyrosine and a phenanthridine derivative, was synthesized. Binding of the dipeptide to a series of polynucleotides including ct-DNA, poly A - poly U, poly (dAdT)2, poly dG - poly dC and poly (dGdC)2 was investigated by thermal denaturation experiments, fluorescence spectroscopy and circular dichroism. Thermal denaturation experiments indicated that dipeptide 4 at pH 5.0, when phenanthridine is protonated, stabilizes ds-DNA, whereas it destabilizes ds-RNA. At pH 7.0, when the phenanthridine is not protonated, effects of 4 to the polynucleotide melting temperatures are negligible. At pH 5.0, dipeptide 4 stabilized DNA double helices, and the changes in the CD spectra suggest different modes of binding to ds-DNA, most likely the intercalation to poly dG- poly dC and non-specific binding in grooves of other DNA polynucleotides. At variance to ds-DNA, addition of 4 destabilized ds-RNA against thermal denaturation and CD results suggest that addition of 4 probably induced dissociation of ds-RNA into ss-RNA strands due to preferred binding to ss-RNA. Thus, 4 is among very rare small molecules that stabilize ds-DNA but destabilize ds-RNA. However, fluorescence titrations with all polynucleotides at both pH values gave similar binding affinity (log Ka ≈ 5), indicating nonselective binding. Preliminary photochemical experiments suggest that dipeptide 4 reacts in the photochemical reaction, which affects polynucleotides chirality, presumably via quinone methide intermediates that alkylate DNA.

Studies on the Melting of DNA

1974 ◽  
Vol 29 (3-4) ◽  
pp. 130-132
Author(s):  
Gokul Chandra Das
Keyword(s):  
Ionic Strength ◽  
Melting Temperature ◽  
Thermal Denaturation ◽  
Spectrophotometric Methods ◽  
Melting Temperatures

Abstract The thermal denaturation of the native DNA in solvents of varying salt concentrations was studied by viscometric and spectrophotometric methods. It was observed that within the molarity range of 0.02 ᴍ to 0.3 ᴍ, the melting temperatures obtained by the two independent methods agreed well, but that at lower ionic strength the agreement was not satisfactory. Both the visco­metric and the spectrophotometric measurements showed an increase of the melting temperature with increasing counterion concentration and a levelling off effect in the neighbourhood of 0.3 ᴍ.

ASP53, a thermostable protein from Acacia erioloba seeds that protects target proteins against thermal denaturation

2007 ◽  
Vol 34 (2) ◽  
pp. 139 ◽  
Author(s):  
Linda Mtwisha ◽  
Jill M. Farrant ◽  
Wolf Brandt ◽  
Caswell Hlongwane ◽  
George G. Lindsey
Keyword(s):  
Secondary Structure ◽  
Thermal Denaturation ◽  
Storage Proteins ◽  
Seed Storage ◽  
Seed Storage Proteins ◽  
Temperature Dependent ◽  
Alcohol Dehydrogenase Activity ◽  
Thermostable Protein ◽  
Three Stages ◽  
Mature Seeds

ASP53, a 53 kDa heat soluble protein, was identified as the most abundant protein in the mature seeds of Acacia erioloba E.Mey. Immunocytochemistry showed that ASP53 was present in the vacuoles and cell walls of the axes and cotyledons of mature seeds and disappeared coincident with loss of desiccation tolerance. The sequence of the ASP53 transcript was determined and found to be homologous to the double cupin domain-containing vicilin class of seed storage proteins. Mature seeds survived heating to 60°C and this may be facilitated by the presence of ASP53. Circular dichroism spectroscopy demonstrated that the protein displayed defined secondary structure, which was maintained even at high temperature. ASP53 was found to inhibit all three stages of protein thermal denaturation. ASP53 decreased the rate of loss of alcohol dehydrogenase activity at 55°C, decreased the rate of temperature-dependent loss of secondary structure of haemoglobin and completely inhibited the temperature-dependent aggregation of egg white protein.

Application of Taguchi Method in the Optimization of Antioxidant Activity for Australian Tea Tree

2014 ◽  
Vol 595 ◽  
pp. 253-257
Author(s):  
Shu Wen Wang ◽  
Te Li Su ◽  
Jhe Yu Ye ◽  
Lin Lo
Keyword(s):  
Antioxidant Activity ◽  
Solvent Extraction ◽  
Taguchi Method ◽  
Experimental Parameter ◽  
General Term ◽  
Quality Data ◽  
Key Factors ◽  
Experimental Procedure ◽  
Tea Tree ◽  
Experimental Parameters

Tea tree is the general term for many species of melaleuca plants. The essential oil or liquid extracted from Australian tea tree can be added to cosmetics and care products. The experimental parameters of solvent extraction are the key factors influencing the antioxidant activity of Australian tea tree. This study used the orthogonal array of the Taguchi method to plan the experiment. The quality data obtained from experiment were analyzed by the factor effects of the Taguchi method to find the significant experimental parameters influencing the antioxidant activity of Australian tea tree, and obtain the optimal experimental parameter combination of solvent extraction for antioxidant activity. Finally, the reliability of the experiment was validated by a confirmation experiment. According to the experimental results, this method was applicable to the optimization problem of the experimental procedure. The optimization can be implemented for objective numerical analysis operation, saving on experimental consumables, time and cost.

scholarly journals Interaction between Chromosomal Protein HMGB1 and DNA Studied by DNA-Melting Analysis

2014 ◽  
Vol 2014 ◽  
pp. 1-5 ◽  
Author(s):  
Elena V. Chikhirzhina ◽  
Starkova J. Tatiana ◽  
Alexander M. Polyanichko
Keyword(s):  
Circular Dichroism ◽  
Thermal Denaturation ◽  
Circular Dichroism Spectroscopy ◽  
Dna Melting ◽  
Chromosomal Protein ◽  
Melting Temperatures ◽  
Melting Analysis ◽  
Circular Dichroïsm ◽  
Nonhistone Chromosomal Protein ◽  
Biphasic Process

Interaction of HMGB1 nonhistone chromosomal protein with DNA was studied using circular dichroism spectroscopy and thermal denaturation of DNA. Melting DNA in the complex was shown to be a biphasic process. The characteristic melting temperatures of unbound DNA and the DNA bound to HMGB1 in 0.25 mM EDTA solutions were found to beTmI=44.0±0.5°C andTmII=62.0±1°C, respectively. It was shown that the binding of the HMGB1 molecule affects the melting of the DNA region approximately 30 b.p. long.

scholarly journals Structural Characterization of the C2 Domains of Classical Isozymes of Protein Kinase C and Novel Protein Kinase Cε by using Infrared Spectroscopy

2003 ◽  
Vol 17 (2-3) ◽  
pp. 399-416
Author(s):  
Senena Corbalán-García ◽  
Josefa García-García ◽  
M. Susana Sánchez-Carrillo ◽  
Juan C. Gómez-Fernández
Keyword(s):  
Thermal Stability ◽  
Protein Kinase ◽  
Secondary Structure ◽  
Phosphatidic Acid ◽  
Bound States ◽  
Thermal Denaturation ◽  
Spectroscopic Studies ◽  
Lipid Binding ◽  
C2 Domain ◽  
Β Sheet

The amide I regions in the original infrared spectra of PKCα-C2 in the Ca2+-free and Ca2+-bound states are both consistent with a predominantly β-sheet secondary structure. Spectroscopic studies of the thermal denaturation revealed that for the PKCα-C2 domain alone the secondary structure abruptly changed at 50°C. While in the presence of Ca2+, the thermal stability of the protein increased considerably. Phosphatidic acid binding to the PKCα-C2 domain was characterized, and the lipid–protein binding becoming Ca2+-independent when 100 mol% phosphatidic acid vesicles was used. The effect of lipid binding on secondary structure and thermal stability was also studied. In addition, the secondary structure of the C2 domain from the novel PKCε was also determined by IR spectroscopy and β-sheet was seen to be the major structural component. Spectroscopic studies of the thermal denaturation in D2O showed a broadening in the amide I′band starting at 45°C. Phosphatidic acid containing vesicles were used to characterize the effect of lipid binding on the secondary structure. It was observed through thermal stability experiments that the secondary structure did not change upon lipid binding and the protein stability was very high with no significant changes occurring in the secondary structure after heating.

scholarly journals Antha-guided Automation of Darwin Assembly for the Construction of Bespoke Gene Libraries

2019 ◽  
Author(s):  
P. Handal Marquez ◽  
M. Koch ◽  
D. Kestemont ◽  
S. Arangundy-Franklin ◽  
V. B. Pinheiro
Keyword(s):  
Saturation Mutagenesis ◽  
Liquid Handling ◽  
Assembly Method ◽  
Amplicon Length ◽  
Hands On ◽  
Experimental Parameters ◽  
Gene Libraries ◽  
Fast Reconstruction ◽  
Protein Libraries

AbstractProtein engineering through directed evolution facilitates the screening and characterization of protein libraries. Efficient and effective methods for multiple site-saturation mutagenesis, such as Darwin Assembly, can accelerate the sampling of relevant sequence space and the identification of variants with desired functionalities. Here, we present the automation of the Darwin Assembly method, using a Gilson PIPETMAX™ liquid handling platform under the control of the Antha software platform, which resulted in the accelerated construction of complex, multiplexed gene libraries with minimal hands-on time and error-free, while maintaining flexibility over experimental parameters through a graphical user interface rather than requiring user-driven library-dependent programming of the liquid handling platform. We also present an approach for barcoding libraries that overcomes amplicon length limitations in next generation sequencing and enables fast reconstruction of library reads.

scholarly journals Mechanism of thermal denaturation of maltodextrin phosphorylase from Escherichia coli

2000 ◽  
Vol 346 (2) ◽  
pp. 255-263 ◽  
Author(s):  
Richard GRIEßLER ◽  
Sabato D'AURIA ◽  
Reinhard SCHINZEL ◽  
Fabio TANFANI ◽  
Bernd NIDETZKY
Keyword(s):  
Escherichia Coli ◽  
Secondary Structure ◽  
Conformational Changes ◽  
Active Site ◽  
Thermal Denaturation ◽  
Hydrophobic Interactions ◽  
Wild Type ◽  
Reversible Inactivation ◽  
The Stability ◽  
Maltodextrin Phosphorylase

Maltodextrin phosphorylase from Escherichia coli (MalP) is a dimeric protein in which each ≈ 90-kDa subunit contains active-site pyridoxal 5ʹ-phosphate. To unravel factors contributing to the stability of MalP, thermal denaturations of wild-type MalP and a thermostable active-site mutant (Asn-133 → Ala) were compared by monitoring enzyme activity, cofactor dissociation, secondary structure content and aggregation. Small structural transitions of MalP are shown by Fourier-transform infrared spectroscopy to take place at ≈ 45 °C. They are manifested by slight increases in unordered structure and 1H/2H exchange, and reflect reversible inactivation of MalP. Aggregation of the MalP dimer is triggered by these conformational changes and starts at ≈ 45 °C without prior release into solution of pyridoxal 5ʹ-phosphate. It is driven by electrostatic rather than hydrophobic interactions between MalP dimers, and leads to irreversible inactivation of the enzyme. Aggregation is inhibited efficiently and specifically by oxyanions such as phosphate, and AMP which therefore, stabilize MalP against the irreversible denaturation step at 45 °C. Melting of the secondary structure in soluble and aggregated MalP takes place at much higher temperatures of approx. 58 and 67 °C, respectively. Replacement of Asn-133 by Ala does not change the mechanism of thermal denaturation, but leads to a shift of the entire pathway to a ≈ 15 °C higher value on the temperature scale. Apart from greater stability, the Asn-133 → Ala mutant shows a 2-fold smaller turnover number and a 4.6-fold smaller energy of activation than wild-type MalP, probably indicating that the site-specific replacement of Asn-133 brings about a greater rigidity of the active-site environment of the enzyme. A structure-based model is proposed which explains the stabilizing interaction between MalP and oxyanions, or AMP.

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