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Peptide Design Introduction

HongTide gained extensive experience on peptide synthesis, here we list our suggestions on peptide design according to the experience we gained in our research.
Peptide Application Introduction
With the development of biotechnology, peptides have been widely used in many areas including:

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

Principle of Peptide Design

Design basics I：charge

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

Design basics II: hydrophobicity

Hydrophobicity peptides are:

>5 AA long

Containing >50% hydrophobic amino acids

Solution: Avoid hydrophobicity by replacing non-essential hydrophobic amino acids with charged or polar residues.

Design basics III: length

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.

Design basics IV：purity selection

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:

Applications

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

Recommended Purity

Crude peptide

70% and >75%

>80%，>85% and >90%

>95%

>98%

Advanced Design Strategy: Sequence Satability

The reasons and solutions to cause peptide unstable in peptide production:

  • Design Constraint
    Susceptible amino acids & Sequence
    Strategies to increase stability
  • Cyclization
    Asp-Gly
    N-terminal Gln
    Substitute Asp with other amino acids
    Acetylate N-terminal Gln
  • Secondary structure formation
    Multiple Glu, Ile, Leu, Phe, Thr, Tyr, Val
    Asp for Glu
    Ser for Thr
    Pro or Gly every third residue
  • Oxidation
    Cys，Met
    Ser with Cys
    Nle with Met
  • Hydrolysis
    Asp-Gly; Asp-Pro; Asp-Ser
    Substitute Asp with other amino acids
  • Coloration
    Trp
    Substitute Trp with other amino acids

Protease/peptidase degradation:

  • 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.

Renal Clearance:

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.

Why Peptgide Asssay Failed?

Reasons of peptide assay failure: 1) Oxidation; 2) Poor solubility; 3) Improper storage conditions; 4) TFA counter-ion contamination; 5) Biological contamination.

1）Oxidation

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.

2）Poor Solubility

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.

3）Improper storage conditions

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.

''Do's'' and ''Dont's''of Peptide Handling：

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;

DON'T store peptides long-term in solution;

DON'T repeatedly open the stock peptide vial;

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;

4） TFA Counter-ion 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;

5） Biological Contamination

Symptoms:

Endotoxin contamination;

Unwanted immune reactions;

Affected Sequences: All sequences.

Solutions:

Endotoxin removal;

Open peptide vials only in a sterile hood.

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