Silk-Cellulose Acetate Biocomposite Materials Regenerated from Ionic Liquid

The novel use of ionic liquid as a solvent for biodegradable and natural organic biomaterials has increasingly sparked interest in the biomedical field. As compared to more volatile traditional solvents that rapidly degrade the protein molecular weight, the capability of polysaccharides and proteins to dissolve seamlessly in ionic liquid and form fine and tunable biomaterials after regeneration is the key interest of this study. Here, a blended system consisting of Bombyx Mori silk fibroin protein and a cellulose derivative, cellulose acetate (CA), in the ionic liquid 1-ethyl-3-methylimidazolium acetate (EMIMAc) was regenerated and underwent characterization to understand the structure and physical properties of the films. The change in the morphology of the biocomposites (by scanning electron microscope, SEM) and their secondary structure analysis (by Fourier-transform infrared spectroscopy, FTIR) showed that the samples underwent a wavering conformational change on a microscopic level, resulting in strong interactions and changes in their crystalline structures such as the CA crystalline and silk beta-pleated sheets once the different ratios were applied. Differential scanning calorimetry (DSC) results demonstrated that strong molecular interactions were generated between CA and silk chains, providing the blended films lower glass transitions than those of the pure silk or cellulose acetate. All films that were blended had higher thermal stability than the pure cellulose acetate sample but presented gradual changes amongst the changing of ratios, as demonstrated by thermogravimetric analysis (TGA). This study provides the basis for the comprehension of the protein-polysaccharide composites for various biomedical applications.

In silico recombinant plasmid design of pHA171 with phdABCD insertion for ethidium bromide degradation

Background: Ethidium bromide is a common reagent that is used in nucleic acid staining. However, ethidium bromide has toxic and carcinogenic properties that are harmful to the environment. Phenanthrene dioxygenase (encoded by phdA, phdB, phdC, and phdD genes) in Nocardioides sp. KP7 can oxidize the phenanthridine structure aim to eliminate carcinogenic properties. Objective: This study aims to visualize and predict the structure, active site, and characteristics of the phenanthrene dioxygenase using bioinformatics tools. Methods: Plasmid design were prepared by inserting genes of interest phdA, phdB, phdC, and phdD from the NCBI database. Furthermore, several protein analysis tools were used for structure visualization, active site enzyme improvement, and protein characteristic of phenanthrene dioxygenase. Results: The prediction results found that phenanthrene dioxygenase reacts with the ethidium bromide substrate through the interaction of Fe3+ ions with water. The solubility level of phenanthrene dioxygenase protein is 0.404, suggesting that the protein has low solubility. The protein isoelectric point (pI) is between 5.17 to 5.36, and the protein molecular weight is 121.143 kDa. Conclusion: In silico analysis has supported that recombinant plasmid met characteristics for the construct which consists of gene interest and protein library.

The Penn State Protein Ladder system for inexpensive protein molecular weight markers

We have created the Penn State Protein Ladder system to produce protein molecular weight markers easily and inexpensively (less than a penny a lane). The system includes plasmids which express 10, 15, 20, 30, 40, 50, 60, 80 and 100 kD proteins in E. coli. Each protein migrates appropriately on SDS-PAGE gels, is expressed at very high levels (10–50 mg per liter of culture), is easy to purify via histidine tags and can be detected directly on Western blots via engineered immunoglobulin binding domains. We have also constructed plasmids to express 150 and 250 kD proteins. For more efficient production, we have created two polycistronic expression vectors which coexpress the 10, 30, 50, 100 kD proteins or the 20, 40, 60, 80 kD proteins. 50 ml of culture is sufficient to produce 20,000 lanes of individual ladder protein or 3750 lanes of each set of coexpressed ladder proteins. These Penn State Protein Ladder expression plasmids also constitute useful reagents for teaching laboratories to demonstrate recombinant expression in E. coli and affinity protein purification, and to research laboratories desiring positive controls for recombinant protein expression and purification.

Critical Aspects to Produce Low-Cost Protein Molecular Weight Marker: A Review

Proteomic studies usually begin with characterizing protein profile, like the molecular weight of protein, which can be done by the SDS-PAGE technique followed by Western Blot. These methods need a standard protein called molecular weight marker (MWM). In this review, the important, basic aspects of either recombinant or native MWM production was discussed, including the type and effect of dyes can be used for preparing a prestained MWM. Moreover, buffer and polyols used in the formulation can also affect the quality and stability. Also, another adjuvant may be needed to increase the robustness of MWM to lower the risk of protease contamination that can breakdown the protein inside the MWM during storage. Understanding those critical aspects will help to produce/formulate a good quality of MWM.

Signal metrics analysis of oscillatory patterns in bacterial multi-omic networks

Motivation One of the branches of Systems Biology is focused on a deep understanding of underlying regulatory networks through the analysis of the biomolecules oscillations and their interplay. Synthetic Biology exploits gene or/and protein regulatory networks towards the design of oscillatory networks for producing useful compounds. Therefore, at different levels of application and for different purposes, the study of biomolecular oscillations can lead to different clues about the mechanisms underlying living cells. It is known that network-level interactions involve more than one type of biomolecule as well as biological processes operating at multiple omic levels. Combining network/pathway-level information with genetic information it is possible to describe well-understood or unknown bacterial mechanisms and organism-specific dynamics. Results Following the methodologies used in signal processing and communication engineering, a methodology is introduced to identify and quantify the extent of multi-omic oscillations. These are due to the process of multi-omic integration and depend on the gene positions on the chromosome. Ad hoc signal metrics are designed to allow further biotechnological explanations and provide important clues about the oscillatory nature of the pathways and their regulatory circuits. Our algorithms designed for the analysis of multi-omic signals are tested and validated on 11 different bacteria for thousands of multi-omic signals perturbed at the network level by different experimental conditions. Information on the order of genes, codon usage, gene expression and protein molecular weight is integrated at three different functional levels. Oscillations show interesting evidence that network-level multi-omic signals present a synchronized response to perturbations and evolutionary relations along taxa. Availability and implementation The algorithms, the code (in language R), the tool, the pipeline and the whole dataset of multi-omic signal metrics are available at: https://github.com/lodeguns/Multi-omicSignals. Supplementary information Supplementary data are available at Bioinformatics online.

Evaluation of Strategies for Decreasing Blood Glucose Using Albuminbinding Domain

Objective: Frequent administrations for DPPIV-resistant GLP-1 analogs are necessary to maintain the blood concentrations due to the short half-life of less than 5 minutes. However, most delivery systems that possess the ability of sustainable release of GLP-1 have drawbacks such as low yield, high cost and undesirable side effects. Therefore, we aimed to prepare a simple and efficient delivery system that could be feasibly applied to reduce blood glucose. Methods: A novel GLP-1 delivery system (GLP-1-ELPs-SA) was prepared and characterized by circular dichroism. Furthermore, the activity and property of GLP-1-ELPs-SA were evaluated in vitro and in vivo. Results: GLP-1-ELPs-SA are easily expressed in E. coli in a soluble formulation and purified through the inverse transition cycle. GLP-1-ELPs-SA spontaneously generated depot under physiological conditions. GLP-1-ELPs-SA was also found to be dispersed in the blood vessels from the depot and showed a high affinity to bind with mice (C57BL/6J) albumin, which shows that GLP-1-ELPs-SA has a long circulation time in vivo. Conclusions: Our delivery system could markedly decrease the clearance of recombinant proteins based on serum albumin, without substantially increasing the protein molecular weight and remarkably reducing the blood glucose within 120 h.

Incorrect Molecular Weights due to inaccurate Prestained Protein Molecular Weight Markers that are used for Gel Electrophoresis and Western Blotting

ABSTRACTThe most widely used Western blotting protein standards are prestained proteins of known molecular mass (kDa). They are also utilized for sodium dodecyl sulphate (SDS) Polyacrylamide Gel Electrophoresis (PAGE) to determine the molecular mass of proteins separated by electrophoresis. The objective of this study was to assess the reliability of different commercially available protein standards in predicting accurate protein molecular weights. We performed this experiment by running Criterion TGX gels with five prestained protein standards (Thermo Fisher SeeBlue Plus 2, Bio-Rad Precision Plus Protein Dual-color, Thermo Fisher Spectra Multi-color, Novex-Sharp Pre-stained, and Invitrogen iBright Pre-Stained). To evaluate their accuracy, we utilized highly purified Bovine Serum Albumin (BSA, 66.44 kDa) and Cytochrome C (Cyto C, 11.62 kDa). We also made use of the dimers of BSA (132.88 kDa) and Cyt C (23.24 kDa) that are present on SDS-PAGE gels. Our results suggest that three of the standards were less accurate at higher molecular masses with the iBright marker having the highest error in determining the expected 132.88 kDa molecular weight. The SeeBlue Plus 2 was accurate at identifying the 132.88 kDa molecular weight protein band but was less reliable for the three other lower molecular weight proteins. These findings have significant implications for the determination of protein masses because researchers rely on these standards to evaluate the molecular masses of their protein(s). We suggest that at least two different protein standards should be initially used in electrophoresis gels and for Western blotting in order to get accurate protein molecular weight results.