Look Who’s Talking

Intraspecies pheromone signaling regulated by proteases is critical for fungi procreation.  The fungal aspartyl protease Bar1 was shown to have unique substrate specificity of important implications in fungal evolution.  LifeTein synthesized substrate peptides of Bar1, dual-tagged with DABCYL and EDANS, from the sequence of α pheromone, the native target of Bar1. Referred as internally quenched or IQ peptides, they were used in fluorescence resonance energy transfer (FRET) assays to study the enzyme kinetics of Bar1.

LifeTein’s Internally Quenched Peptides

mBio 6(6):e01604-15. doi:10.1128/mBio.01604-15. Evolutionary selection on barrier activity: Bar1 is an aspartyl protease with novel substrate specificity.

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Peptide Applications

Peptides can be used in a wide variety of research applications:

Anti-microbial Peptides

81 oligopeptides were synthesized by LifeTein and tested for inhibition of Enterococcus faecalis V583. Three peptides were found to inhibit V583. The peptide (NH2-VAVLVLGA-COOH) possessed activity in picomolar concentrations, being >10^6 -fold more active than the only other two and showing inhibitory activity. Pheromone killing of multidrug-resistant Enterococcus faecalis V583 by native commensal strains, PNAS, 2015 The fungal pathogen causes the skin disease for amphibians. Use of a potent antibiotic cocktail dramatically reduced culturable skin bacteria within 48 h. The synthetic peptides by LifeTein were used to reduce the skin bacteria. SSkin bacteria provide early protection for newly metamorphosed southern leopard frogs (Rana sphenocephala) against the frog-killing fungus, Batrachochytrium dendrobatidis, Biological Conservation, 2015

Anti-inflammatory Peptides

Anti-inflammatory peptides were isolated from alcalase hydrolysates out of tuna cooking juice by-product. Synthetic peptides from LifeTein were used to confirm the inhibitory anti-inflammatory activity. The amino acid sequences of the two anti-inflammatory peptides isolated from AH hydrolysates were Pro-Arg-Arg-Thr-Arg-Met-Met-Asn-Gly-Gly-Arg (1543.8 Da) and Met-Gly-Pro-Ala-Met-Met-Arg-Thr-Met-Pro-Gly (1211.5 Da).

Epitope Mapping

Peptide scanning involves the chemical synthesis of overlapping peptides covering the antigen sequence targeted by the investigated antibodies. Peptide truncations are used to further narrow down the epitope sequence and mutagenesis of each amino acid such as alanine substitution can also indicate the binding affinity. Cross-reactive epitopes were found in Borrelia burgdorferi p66. Cross-reactive epitopes in Borrelia burgdorferi p66, Clinical and Vaccine Immunology, 2015

Cell Penetrating Peptides and Scrambled Peptides

The CD81 peptides tagged with cell penetrating peptide RRRRRRR were used for the binding assay. The synthetic peptides from LifeTein were used to investigate the role of CD81 in the regulation of defense mechanisms against microbial infections. The scrambled peptides, RRRRRRR- CCGIRNSSVY, were used as the negative control for the study. CD81 Controls Immunity to Listeria Infection through Rac-Dependent Inhibition of Proinflammatory Mediator Release and Activation of Cytotoxic T Cells, The Journal of Immunology, 2015

Receptor Binding Study

His-tagged GLP-1 (7-36), glucagon, and gastric inhibitory polypeptides (GIP) by LifeTein were used to study GLP-1 receptor signaling regulation. The GLP-1 peptides bind specifically with lipids but not that of exendin 4.The His-Tagged GLP-1 were used for the binding reaction. The free peptide was captured by Cu++-NTA resin. The results indicated that His-tagged GLP-1 peptide binds to OEA in a dose-dependent and saturable way. Modulation of Glucagon-like Peptide (GLP)-1 Potency by Endocannabinoid-like Lipids Represents A Novel Mode of Regulating GLP-1 Receptor Signaling. Journal of Biological Chemistry, 2015

Antibody Blocking Peptides

Peptides can be used as blocking peptides for the competition assay. The excess of blocking peptides (20:1 peptide: antibody ratio) from LifeTein were mixed with antibodies. The antibody was neutralized in this way by incubating with an excess of peptide that corresponds to the epitope recognized by the antibody. The neutralized antibody is then used side-by-side with the antibody alone, and the results are compared. Whole Exome Sequencing Reveals ZNF408 as a New Gene Associated With Autosomal Recessive Retinitis Pigmentosa with Vitreal Alterations, Human Molecular Genetics, 2015

Protein-Protein Interactions

The B-cell lymphoma 2 (Bcl-2) peptides were biotinylated at N terminus for the protein-protein interactions. The biotin-BH4-Bcl-XL peptide and the scrambled peptide were immobilized on different channels of a streptavidin-coated sensor chip. Studies showed that Bcl-XL bound to the central domain of RyR3 via its BH4 domain. Further analysis of a mutated peptide at a specific site Lys87 showed a reduced binding affinity. These data suggest that BH4 domain and its specific site of Lys87 contributes to the interaction. Ryanodine receptors are targeted by anti-apoptotic Bcl-XL involving its BH4 domain and Lys87 from its BH3 domain, Nature Scientific Reports, 2015

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LifeTein Peptides Used for Pulldown Assay

LifeTein synthesized multiple peptides for the pulldown experiments. The peptides were synthesized with biotin on the N terminus and an aminohexanoic acid linker. Two types of peptides were designed: peptide without acetylation and peptide with multiple lysines (up to five sites) were acetylated in the synthetic peptides.

Pulldown Experiments With LiteTein Peptides

These peptides were bound to streptavidin-agarose beads and used for pulldown experiments using cell lysates.  Peptides, streptavidin agarose beads, and cell lysates were permitted to bind for overnight at 4 °C,  then beads were pelleted, washed five times using spin columns, and proteins were eluted in sample buffer and analyzed by SDS-PAGE and immunoblotting. All synthetic peptides had an N-terminal biotin. After incubation, peptides were spotted on nitrocellulose membranes and immunoblotted to detect acetylated Lysine (AcK) or total biotinylated peptide. Data is shown below.
Acetylated lysine peptide

Dot blot for biotin peptide with pulldown assay

Reference: Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-Responsive Intracellular Compartment, The Journal of Biological Chemistry, January 5, 2015, doi: 10.1074/jbc.M114.603977 jbc.M114.603977.

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