Protein and Peptide Letters - Volume 25, Issue 11, 2018

Introduction: The giant muscular proteins titin and obscurin bind to each other at the Zdisk during muscle development. This binding event is mediated through two domains from each protein: ZIg9/10 from titin and Ig58/59 from obscurin. This interaction helps stabilize and organize the sarcomere; ablation of this binding leads to muscular dystrophy. Objective: Here we solve the high-resolution solution structure of titin ZIg10 and further delineate which sections of titin bind to obscurin. Materials and Methods: Solution NMR, Circular Dichroism, and SEC-MALS were used to biophysically characterize the titin domains involved in this titin-obscurin interaction. Results and Conclusion: We present the high-resolution solution structure of titin ZIg10. Additionally, we show that titin ZIg9 drives the titin-obscurin interaction, while ZIg10 does not actively participate in the titin-obscurin interaction but instead acts to stabilize ZIg9.

Background: Cryptochrome is a flavin-binding blue-light photoreceptor that functions in growth and development in plants, the circadian clock in animals and navigation in birds. However, a lack of purified cryptochrome has hindered studies of the structure and function of this protein. In this study, we obtained a substantial amount of the Columbia livia Cryptochrome1 (ClCry1) protein by using a prokaryotic expression system. In addition, we performed comprehensive experiments to assess the influence of several factors on the purification and yield of ClCry1, such as the inducer that was used, temperature, duration of expression and type of growth medium. These assays clearly indicated that such factors influenced the purification and yield of ClCry1. Moreover, Flavin Adenine Dinucleotide (FAD) was added during expression and purification of ClCry1, which resulted in production of large amounts of ClCry1 protein with the FAD cofactor from the Escherichia coli (E. coli) heterologous expression system. We believe that this study provides a novel avenue to acquire large amounts of ClCry1 that contains FAD and lays the foundation for studies of the geomagnetic navigation mechanism of Aves. Objective: In this article, our motivation is to sufficiently acquire functional ClCry1 protein. Method: In this article, we performed series of experiments to optimize the yields of ClCry1 protein expression by conducting with expression-vectors, variable inducers, temperatures, medias and durations of induction, which also identified the most appropriate conditions for obtaining functional ClCry1. Moreover, we identified a solution for the FAD abscission of ClCry1 by adding additional FAD into the dialysis buffer during the purification. Results: Following our performed series of experiments, we assessed several crucial parameters, such as inducer, temperature, duration of induction, culture medium and recombinant expression vector. The highest yields of ClCry1 were observed with 0.01 mM IPTG and expressing for 8 h with pET21a-ClCry1 as recombinant expression vectors. Conclusion: We demonstrated the feasibility of heterologous expression of ClCry1 in E. coli. In addition, we identified a solution for the low yield and FAD abscission of ClCry1 by conducting several experiments with variable inducers, temperatures, medias and durations of induction, which also identified the most appropriate conditions for obtaining functional ClCry1. Moreover, the typical yield was approximately 6 mg of ClCry1 from 2-liter culture, and 50% of the final protein contained the FAD cofactor. These results strongly suggest that our expression strategy is useful.

Background: Human Interleukin 37 (IL37), a unique anti-inflammatory cytokine of IL1 family member, plays critical roles in innate and adaptive immunity and inflammation. Objective: Preparation of high purity and tag-removed recombinant IL37 protein (rIL37) is critical for its clinical application. Method: In this study, we constructed an N-terminal cleavable GST-fused IL37 expression vector for recombinant expression. Results: Subsequent to transformation and optimization of the induction temperature, the soluble expression level of rIL37 was 306.5 mg/L of culture medium at 18 °C induction in Escherichia coli. Meanwhile, rIL37 was digested on beads by GST-HRV3C protease during GST affinity chromatography. After further purification, the purity of rIL37 was higher than 99 %. Finally, the antiinflammatory activity of tag-removed protein was verified by the results showing that rIL37 suppressed IL1β production in PBMCs. Conclusion: This work presents a protocol to produce high purity and tag-removed rIL37 with antiinflammatory activity, which provides the firm basis for advancing clinical application in human IL37-related inflammatory diseases.

Background: Cellulose, being the most abundant biopolymer found in nature, can be utilized for bioethanol production to cater the future energy needs. Due to increased usage of fossil fuel it has been predicted that fossil fuel reserves may be depleted by year 2050. These concerns need serious attention and focus should be diverted to renewable fuels that are based on natural resources. Cellulases including exoglucanase (cellobiohydrolases) are the key enzymes that are produced by cellulolytic micro-organisms for the biodegradation of natural resource (cellulose) into fermentable reducing sugars. Many members of genus Clostridium possess supramolecular structures known as cellulosomes which contain various cellulases. Cellulase are composed of catalytic subunits that include endoglucanase, β-glucosidase and cellobiohydrolases which concurrently can catalyse and subsequently convert cellulose into glucose and other sugars. After the action of cellulases, the sugars can be conveniently converted into bioethanol. Objective: In the present study, characterization of a thermostable cellobiohydrolase enzyme from Thermotoga petrophila was carried out. The main purpose of this study is the utilization of thermostable cellobiohydrolase along with other cellulases in the process of saccharification of the cellulosic biomass to produce fermentable sugars that could in turn be converted into bioethanol which is the fuel of the future. Method: In this article, we propose a framework for achieving our a forementioned object. We started with the cloning of thermophilic cellobiohydrolase gene in mesophilic hosts to ease enzyme production. After cloning of cellobiohydrolase gene, submerged fermentation was performed for intracellular enzyme production. Microbial pellet obtained after centrifugation was sonicated and subjected to ammonium sulphate precipitation. The fraction obtained was purified to isoelectric homogeneity through ion exchange chromatography. Finally SDS analysis of purified cellobiohydrolase was carried out alongwith its characterization, kinetics and thermodynamics studies. Results: Purification fold of 4.05 was obtained along with enzyme activity and specific activity of 11.5 U ml-1 min-1 and 66.5 U mg-1, respectively. The molecular mass of purified recombinant enzyme was 37 kDa as calculated by means of SDS-PAGE analysis. The enzyme showed 50% residual activity at 90°C and also at a wide pH range of 4-10. The enzyme retained its activity in the presence of most of the metal ions except Fe+2, Hg+2 and Pb+2. EDTA has an inhibitory effect on the function of the enzyme. The catalytic activity of the enzyme was maintained in the presence of the organic solvents. The enzyme had a Km and Vmax of 4.6 mM and 25.64±1.87 μM min-1 for PNP-β- D-cellobioside under optimal conditions. Conclusion: The present study demonstrated that cellobiohydrolase produced from Thermotoga petrophila can be employed in many industries like paper and pulp and food processing. Most recent application of the cellobiohydrolases is their utilization in the production of bioethanol.

Background: Local classification models were used to establish Quantitative Structure− Activity Relationships (QSARs) of bioactive di−, tri− and tetrapeptides, with their capacity to inhibit Angiotensin Converting Enzyme (ACE). These discrete models can thus predict this activity for other peptides obtained from functional foods. These types of peptides allow some foods to be considered nutraceuticals. Method: A database of 313 molecules of di−, tri− and tetrapeptides was investigated and antihypertensive activities of peptides, expressed as log (1/IC50), were separated into two qualitative classes: low activity (inactive) was associated with experimental values under the 66th percentile and active peptides with values above this threshold. Chemicals were divided into a training set, including 70% of the peptides, and a test set for external validation. Genetic algorithms-variable subset selection coupled with the kNN and N3 local classifiers were applied to select the best subset of molecular descriptors from a pool of 953 Dragon descriptors. Both models were validated on the test peptides. Results: The N3 model turned out to be superior to the kNN model when the classification focused on identifying the most active peptides.

Background: Der p 5 is an important allergen of Dermatophagoides pteronyssinus that plays a key role in allergic airway diseases. Its three dimensional structure (PDB 3MQ1) consists of three anti-parallel α-helices arranged in a helical bundle. Aggregation of Der p5 can modulate its allergenicity. This study aimed to identify the key residues of IgE binding epitopes of Der p 5. Methods: IgE binding epitopes of Der p 5 were characterized as follow. An in silico prediction of the epitope was performed with the help of SEPPA program. We also made a mapping of the epitope by using an overlapping library of peptides that encompass the sequence of mature Der p 5. Finally, an alanine scanning mutagenesis allowed us to define the key residues of the allergen involved in its interaction with IgE. The integrity of the structure of the different protein's mutants was assessed by far UV circular dichroism. Results: The presented data indicate that the major epitope sequence of Der p 5 is 90DRLMQRKDLDIFEQYNLEM108. Residues L98, D99, I100, F101, E102 and Y104 appear to be important for IgE binding. Conclusion: This study highlighted the residues of Der p 5 essential for IgE binding. The identification of the major residues epitope of Der p 5 allergen may participate in the selection and engineering of new hypoallergens used in immunotherapy.