Peptide Design Introduction
Structural Biology
Drug Delivery
Antibody Generation
Drug Discovery
Vaccine Development
Tissue Engineering
NMR
Protein-protein interactions
Enzyme assays
siRNA delivery
Phosphospecific antibodies
Non-commercially available antibodies
Antimicrobials
Cancer (GPCR agonists)
Diabetes (GIP and GLP-1 agonists)
Neurodegenerative disease
HIV
Cancer
Influenza
HPV
Hydrogels
Stem cells
Wound healing
Charge influences:
Solubility
Peptide activity
Attraction to contaminants
Charge is dependent on ionizable groups:
N-term amine, C-term carboxyl
R-groups: Asp, Glu, His, Cys, Tyr, Lys, Arg
Key solubility relationship: pH/pI
pH= pI: minimal solubility, precipitation
pH< pI: net positive charge
pH> pI: net negative charge
Key contamination relationships:
Lys, His, Arg bind TFA, AcOH, HCl
Lys, Arg bind water molecules
Hydrophobicity peptides are:
>5 AA long
Containing >50% hydrophobic amino acids
You can design peptides with any length, but from the positon of peptide synthesis, the difficluty of synthesis will increase as the growth of the peptide length. So we suggest you design the peptide with the length between 15-20 animo acids residues, peptides with < 30 amino acids residues can be synthesized at most conditions, but the success rate to synthesize the peptide with >40 amino acids will be greatly reduced.
HongTide proposes a range of different purity levels to help you make the right choice for your application. Crude peptides are not recommended for biological assays. Crude peptides may contain large amounts of non-peptide impurities such as residual solvents, scavengers from cleavage, TFA and other truncated peptides. TFA cannot be totally removed. Peptides with purity >70% are always used for generating or testing antibodis. Peptides with purity level >85% are usually used in enzymology or biological activity studies. Peptides with purity >95% are excellent for quantitative analysis. We also provide purity peptides with purity >98% in large quantities with commercial applications for our industrial customers. HongTide recommends the following levels of peptide purity for various projects:
Functional screening
Peptide arrays
Antigens for antibody production
Competitive elution chromatography
ELISA standards for measuring antisera titers
Western blotting studies (non-quantitative)
Enzyme-substrate studies (non-quantitative)
Peptide blocking studies (non-quantitative)
Affinity purification
Phosphorylation assays
Protein electrophoresis applications and immunocytochemistry
ELISA standards and RIA protocols (quantitative)
Receptor-ligand interaction studies (quantitative)
In vitro bioassays and in vivo studies
Enzyme studies and blocking assays (quantitative)
NMR studies
Mass spectrometry
Other quantitative assays
SAR Studies
Clinical trials
APIs (Active Pharmaceutical Ingredients)
Commercial products
X-ray crystallography studies
Other sensitive experiments: enzyme-substrate studies, receptor-ligand interaction studies, blocking and competition assays
Crude peptide
70% and >75%
>80%,>85% and >90%
>95%
>98%
Exopeptidases(e.g. aminopeptidases, carboxypeptidases): N-terminal residues correlation:
Longer half-life: Met, Ser, Ala, Thr, Val, or Gly;
Shorter half-life: Phe, Leu, Asp, Lys, or Arg;
Endopeptidases (e.g. trypsin, chymotrypsin, pepsin, elastase)
Susceptible domains: Pro, Glu, Ser, and Thr rich
trategies to reduce degradation: cyclization, acetylation, amidation, D-amino acid replacement, peptoids, hydrocarbon stapling.
Peptides sensitive to renal clearance: 1) Hydrophilic peptides < 25 kDa are susceptible to rapid filtration through the glomeruli of the kidney;
2) Peptides not easily reabsorbed through the renal tubule.
Strategy to decrease renal clearance: 1) conjugation to macromolecules or polymers: polyethylene glycol (PEG), polysialic acid (PSA), hydroxyethylstarch (HES), bovine serum album (BSA);
2) Lipidation.
Symptoms:
Peptide loses activity over time;
Peptide color changes from white to yellow, tan or brown.
Affected Sequences: sequences containing: Cysteine, Tryptophan, Methionine.
Prevention Strategies:
Design optimization: Ser for Cys, Nle for Met;
Peptide protection: 1) Aliquot peptides; 2) Flush peptides with argon gas and store in a tightly sealed container.
Assay Protection: 1) Add reductants: DTT, TCEP, β-mercaptoethanol; 2) Flush buffers with argon gas; 3) Reconstitute buffers and peptide with argon-flushed water in an anaerobic chamber; 4) Perform assays in anaerobic chamber.
Symptoms:
Turbidity or precipitation following peptide dissolution
Affected Sequences: Sequences >5 AA long, and containing >50%Trp, Phe, Tyr, Met, Ile, Leu, Val;
Strategies:
Follow the solubility chart recommendations;
Request solubility testing;
Residue substitutions;
Incorporate of O-acyl bonds;
Incorporate hydrophilic linkers;
For libraries introduce a frame shift.
Symptoms:
Peptide loses activity over time;
yophilized peptide converts from powder to crystal;
Affected Sequences: Sequences containing: Ser ? Thr ? Lys ? Gly ? Arg.
Strategies:
Aliquot peptides;
Store peptides at -20°C and away from light;
Avoid freeze-thaw cycles.
For ALL peptides
For peptides containing Cys, Met, or Trp residues
For peptides containing Asp, Glu, Lys, Arg, or His
For peptides that must be stored in solution
DO aliquot lyophilized peptide according to daily;
DO avoid repeated freeze-thaw cycles;
DO limit peptide exposure to air;
DO purge assay buffers with argon or nitrogen gas;
DO store peptides in tightly capped vials;
DO limit peptide exposure to air;
DO store lyophilized peptides in a desiccator;
DO store peptides in tightly capped vials;
DO avoid repeated freeze-thaw cycles;
DO aliquot your peptide solution according to daily experimental needs;
DO use sterile buffers to dissolve your peptide;
DO filter your peptide using a 0.2 μm filter to remove bacterial contamination;
Symptoms:
Erratic cell or tissue viability, enzyme assay results;
IR spectroscopy data;
Reduced mass spec sensitivity;
Peptide degradation;
Affected Sequences: All sequences, especially those containing positively charged: Lys, His, Arg.
Solutions:
TFA removal service;
Symptoms:
Endotoxin contamination;
Unwanted immune reactions;
Affected Sequences: All sequences.
Solutions:
Endotoxin removal;
Open peptide vials only in a sterile hood.