Peptide sequence design

Peptide sequence design mainly includes the following contents that need to be paid attention to

First, peptide sequence length design:

The purity of peptide synthesis will decrease with the increase of peptide sequence length, especially the sequence with length greater than 30 amino acids, because the peptide synthesis is from C-end to N-end. In the process of amino acid forming peptide bond in each step, the reaction cannot reach 100%, and a incomplete peptide lacking one or two amino acids will be formed. This incomplete peptide is very similar to the target peptide sequence in nature and is not easy to be separated in the purification process, Therefore, this factor should be considered when designing the length of polypeptide sequence.

Second, peptide solubility design:

The side chain of amino acids has the characteristics of hydrophobicity and hydrophilicity. Amino acids can be classified according to the hydrophilicity index. The proportion of hydrophobic amino acids in the polypeptide sequence will affect the solubility of the polypeptide:

Third, peptide amino acid classification:

Hydrophobic polypeptide amino acids (nonpolar): alanine (ALA), phenylalanine (PHE), leucine (Leu), isoleucine (ILE), valine (VAL), methionine (MET), tryptophan (TRP)

Uncharged polypeptide amino acids (polarity): glycine (Gly), proline (pro), asparagine (ASN), glutamine (Gln), serine (SER), threonine (THR), cysteine (Cys), tyrosine (Tyr)

Acidic polypeptide amino acid (polar charged): aspartic acid (ASP), glutamic acid (Glu)

Basic polypeptide amino acids (polar charged): lysine (Lys), arginine (ARG), histidine (his)

It is suggested that the content of hydrophobic amino acids should be kept below 50% of the proportion of polypeptide amino acids, and every five amino acids contain at least one charged amino acid. At physiological pH value, aspartic acid (ASP), glutamic acid (Glu), lysine (Lys) and arginine (ARG) all contain charged side chains, and a single conservative substitution, such as Gly replacing ala or adding polar amino acids at the N or C ends, It can improve the solubility of polypeptide sequence.

Fourth, peptide sequence stability design:

The following design strategies can improve the stability of polypeptide and obtain higher purity and solubility of polypeptide. Different amino acid composition of polypeptide sequence will affect the overall stability, so it needs to be considered in polypeptide sequence design:

1. The sequence contains amino acids such as cysteine (Cys), methionine (MET) and tryptophan (TRP). Due to the sensitivity of oxidation or side reactions, it will affect the high purity of the polypeptide and the overall stability of the polypeptide. When designing the polypeptide sequence, try to reduce the number of these amino acid residues, or choose to replace these amino acid residues. Adding n-leucine can replace methionine (MET), Serine can replace cysteine (Cys). These peptide design strategies can greatly improve the stability of peptides.

2. Instability of N-terminal glutamine (Gln)

When glutamine (Gln) is at the N-terminal, it may be cyclized to pyroglutamic acid when exposed to acidic cleavage conditions. The peptide design strategy of N-terminal acetylation modification of glutamine (Gln) at the N-terminal of polypeptide can be considered, which can greatly improve the stability of polypeptide.

3. When asparagine (ASN) is at the N-terminal, it will also affect peptide synthesis. Asparagine (ASN) has a side chain protective group, which is very difficult to remove at the N-terminal. In peptide sequence design, we can consider the method of extending one amino acid.

4. If multiple proline (pro) are adjacent or proline (pro) and serine (s) are adjacent, the purity of the synthesized polypeptide sequence will be low and many incomplete peptides will be produced. The polypeptide of multiple proline (pro) will undergo CIS trans isomerization in the synthesis, resulting in a significant reduction in the purity of the polypeptide.

5. Beta folding also needs to be considered. It will lead to difficulty in peptide synthesis and can not synthesize long sequences.

Try to avoid the adjacency or duplication of these amino acids in the sequence. They are valine (VAL), isoleucine (ILE), tyrosine (Tyr), phenylalanine (PHE), tryptophan (TRP), leucine (Leu), glutamine (Gln) and threonine (THR). It can be considered to use replacement methods to break, for example, insert a glycine (Gly) or proline (pro) on every three residues, replace glutamine (Gln) with asparagine (ASN), and replace threonine (THR) with serine (SER)