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Bacteriophage Scaffolds for Functional Assembly of Molecules and Nanomaterials
Published in Gilson Khang, Handbook of Intelligent Scaffolds for Tissue Engineering and Regenerative Medicine, 2017
Mi Hwa Oh, Jeong Heon Yu, Moon Young Yang, Yoon Sung Nam
The concept of phage display was first suggested to find epitope-binding regions of antibodies using randomized peptide libraries in 1985.3 Over more than two decades, phage display has become a standard tool for biological screening to identify target peptides in various research fields of molecular biology and drug discovery.24 In phage display, an inserted DNA sequence is fused to an intrinsic viral gene as a part of a viral particle.3 Large peptide libraries displayed on phages are screened in the process referred to as biopanning to select and isolate a target-binding phage. General procedures of biopanning consist of incubation of a phage library with substrates of interest, elimination of unbound phages, and elution and amplification of bound phages. Target-binding phages are gradually enriched by repeated biopanning, each phage is isolated from a plaque assay, and DNA sequencing is performed for each phage to verify a target peptide (Fig. 26.1).4
Targeted proteomic approaches in the context of COVID-19 pandemic
Published in Sanjeeva Srivastava, Multi-Pronged Omics Technologies to Understand COVID-19, 2022
Mehar Un Nissa, Alisha Srivastava, Medha Gayathri J. Pai
Once data acquisition is completed, the experiment results in the generation of .raw files. These raw files can now be imported to Skyline and analyzed. The process of identifying the right peak of the target peptide is called Peak Annotation and is the most crucial part of data analysis of an SRM/PRM experiment. Several parameters should be considered while annotating peaks: Co-elution of transitions: All the transitions of a peptide must co-elute.Intensity of the peak: The most intense peaks must be chosen as they are more probable to be corresponding to the target peptide. Also, the peak should be well distinguished from noise signals, if any.Shape of the peak: The peaks should be forming a proper Gaussian shape. It is usually helpful to look for the characteristic peak shape.Retention time prediction: Skyline can be used to predict the time point at which the target peptide would most likely elute. This predicted retention time can hence be used to identify the peak.Comparing with spectral library: If you have built a spectral library, then that can also be very instrumental in identifying the best peak (and hence the target peptide). A dot product depicts the match of your target peptide spectra with the reference peptide in the library spectra. The greater the dot product, the greater the resemblance between the target peptide and the reference peptide.Using synthetic peptides: Synthetic peptides labeled with a heavy isotope can be used to confirm the peak identified is of the right peak.
Rhodiasolv PolarClean – a greener alternative in solid-phase peptide synthesis
Published in Green Chemistry Letters and Reviews, 2021
Ashish Kumar, Anamika Sharma, Beatriz G. de la Torre, Fernando Albericio
Here we have expanded the Green Solid-Phase Peptide Synthesis (GSPPS) toolbox. PolarClean is a suitable solvent for SPPS for short, hurdle-free peptides. It showed the excellent capacity to dissolve all 20 Fmoc derivatives of the proteinogenic amino acids and most of the coupling reagents and additives. Only HBTU and HATU were not compatible with PolarClean. Despite its high viscosity, this novel solvent showed the good capacity to swell the two resins most commonly used, namely polystyrene and ChemMatrix. The synthesis of model peptides using PolarClean as solvent rendered the target peptide as the major component, thus facilitating purification if required. In the context of green chemistry, we envisage that SPPS and other chemical processes will become ‘à la carte’ in the future, in the sense that more than one green solvent will be required. It might be great for the scale-up production of short peptides. Given this consideration, PolarClean is certain to find its place in the GSPPS toolbox.
Functional expression, purification, and antimicrobial activity of a novel antimicrobial peptide MLH in Escherichia coli
Published in Preparative Biochemistry and Biotechnology, 2018
Guo-Li Gong, Yuan Wei, Zhong-Zhong Wang
We synthetized target gene by SOE-PCR technology through designing the synthesis of three primers’ complementary. The genetic engineering bacterium strain pET-32a-MLH/BL21 (DE3) was successfully constructed by genetic engineering technology. After IPTG induction target peptide (approx. 25 kDa) was successfully expressed in E. coli, SDS-PAGE protein electrophoresis showed that most fusion protein was expressed as insoluble protein exists in inclusion form in E. coli and optimized its fermentation condition. The resulted optimum fermentation condition is 1.0 mM IPTG, induction at 37°C for 4 h. Induced products were purified by Ni2+ column. After dialysis, the heterozygous peptides showed a significant antimicrobial activity on E. coli and S. aureus. The MIC of purified heterozygous peptides was 0.019, 0.025 M, respectively.
Gelation-based visual detection of analytes
Published in Soft Materials, 2019
Wangkhem Paikhomba Singh, Rajkumar Sunil Singh
Zhang and co-workers have also developed a simple visual biosensor for glucose using a somewhat different approach (106). The in situ generated gluconic acid during GOx catalyzed oxidation of (D)-glucose was used to protonate specific target peptide molecules and modulate their gelation behavior (Fig. 6a). The non-gelling peptide containing aspartate anion was protonated to give the gelling aspartic acid counterpart, 54, resulting in a sol-gel transition (Fig. 6b). Similarly, the gelling peptide containing lysine, 55, was protonated giving its non-gelling counterpart which is accompanied by a gel-sol phase change (Fig. 6c).