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Peptide design and solubility

Peptide Design

Peptides are complex biomolecules. Depending on the amino acid composition, each peptide is unique with regard to its chemical and physical properties. Most peptides of biological interest are derived from native proteins. The trans-membrane region of a protein is not usually exposed and should be avoided. Similarly, any region that undergoes post-translational modification (e.g. glycosylation), should also be avoided, since antibodies raised against this sequence may not recognize the modified native protein. Frequently, the derived sequences are altered, generally in the ‘non-essential’, or say less-important, amino acid residues, although the important/essential amino acid residues are not always easy to determine.

Bio-Synthesis routinely assists clients to pinpoint potentially problematic amino acids and modify them to improve peptide assembly, solubility, stability, and antigenicity. Our modification methods include:

  • Amino Acid Substitution – Improve the peptide solubility and stability
  • Chemical Modification – Improve solubility and stability, structure-function studies
  • Spacer Insertion - Improve peptide solubility such as O-linker or PEG linker
  • Peptide Conjugation - Antisera production ligand attachments - chromophores, affinity ligands
  • N- and C-Termini Capping – Avoid introducing extra charge, increase stability
  • N-or C-term Adding Single Cys – For linking or conjugation via maleimide approach

Peptide Solubility

The influence of the amino acid sequence on the chemical and physical characteristics of an individual peptide is vast. Thus, we can only give some general guidelines for solubility prediction:

  • Peptides shorter than 5 residues are usually soluble in water or aqueous buffer, except if the whole sequence consists of hydrophobic amino acids (like W, L, I, F, M, V, Y).
  • Hydrophilic peptides containing >25% charged residues (like D, K, R, H, E) and <25% hydrophobic amino acids are usually soluble in water or aqueous buffer.
  • Hydrophobic peptides containing 50% and more hydrophobic residues may be insoluble or only partly soluble in aqueous solutions. In this case we recommend using organic solvents like DMSO, DMF or acetonitrile.
  • Peptides containing a very high (>75%) proportion of D, E, H, K, N, Q, R, S, T, Y are capable of building intermolecular hydrogen bonds (cross-linking), thus forming gels in aqueous solutions. These peptides should either be treated according to the instructions for hydrophobic peptides or by changing the pH value if possible.

Depending on the peptide sequence, some amino acids or peptide properties directly influence physical and/or chemical behavior of peptides. Among these one can find peptide solubility, peptide stability and peptide charge. Please refer to the tables below for amino acid classifications:

Solubility in aqueous solutions    
Hydrophilic amino acids Intermediate Hydrophobic amino acids
D, E, H, K, N, Q, R, S, T, hydroxy-proline, pyro-glutamic acid C, G A, F, I, L, M, P, V, W, Y,alpha-amino butyric acid, beta-amino alanine, norleucine
Peptide stability    
Subject to oxidation under relatively mild conditions Subject to de-amidation,de-hydration, cyclisation to pGlu Subject to degradation during peptide preparation
C, M N, Q, C-terminal amides, N-terminus Q MW
Peptide charge    
  Positive Negative
  K, R, H, N-terminus of peptide D,E,Y, C-terminus of peptide

The Amino Acids Physical Properties table can be used collectively to analyze an individual amino acid’s effect on a peptide’s overall solubility.  By considering charge, hydropathy, and the presence of free terminal alpha residues, solubility can be predicted with more consistent accuracy.  Peptides should always be solubility tested before large-scale solubility is attempted. 

Keep in mind that charge can also greatly effect solubility.  To minimize any problems, there should be a balance of negative (acid) and positive (basic) amino acids.  If problems are encountered, always add up total pKs (included side chains), and add the appropriate amount of base or acid to the buffer.
Amino Acid Physical Properties

Amino Acids

Code

Hydropathy

Charge

pKa
NH2

pKa
COOH

pK(R)

Solubility

Arginine

R

Hydrophilic

+

9.09

2.18

13.2

71.8

Asparagine

N

Hydrophilic

N

8.8

2.02

 

2.4

Aspartate

D

Hydrophilic

-

9.6

1.88

3.65

0.42

Glutamate

E

Hydrophilic

-

9.67

2.19

4.25

0.72

Glutamine

Q

Hydrophilic

N

9.13

2.17

 

2.6

Lysine

K

Hydrophilic

+

10.28

8.9

2.2

 

Serine

S

Hydrophilic

N

9.15

2.21

 

36.2

Threonine

T

Hydrophilic

N

9.12

2.15

 

Freely

Cysteine

C

Moderate

N

10.78

1.71

8.33

Freely

Histidine

H

Moderate

+

8.97

1.78

6

4.19

Methionine

M

Moderate

N

9.21

2.28

 

5.14

Alanine

A

Hydrophobic

N

9.87

2.35

 

15.8

Valine

V

Hydrophobic

N

9.72

2.29

 

5.6

Glycine

G

Hydrophobic

N

9.6

2.34

 

22.5

Isoleucine

I

Hydrophobic

N

9.76

2.32

 

3.36

Leucine

L

Hydrophobic

N

9.6

2.36

 

2.37

Phenylalanine

F

Hydrophobic

N

9.24

2.58

 

2.7

Proline

P

Hydrophobic

N

10.6

1.99

 

1.54

Tryptophan

W

Hydrophobic

N

9.39

2.38

 

1.06

Tyrosine

Y

Hydrophobic

N

9.11

2.2

10.1

0.038

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