Class Comparison

Differences between Class 1 and Class 2 2A peptides

Two Classes of 2A Peptides

2A peptides fall into two distinct classes based on conserved sequence features.

Class 1

Consensus Sequence

L-L-L-x-x-x-G-D-V-E-x-x-x-x-N-P-G-P

Key Features

• N-terminal: Leucine-rich region (often 3+ leucines)
• Central motif: GDVE (Gly-Asp-Val-Glu)
• C-terminal: NPGP

Representative Peptides

Name	Sequence	Source
T2A	EGRGSLLTCGDVEENPGP	Thosea asigna virus
P2A	ATNFSLLKQAGDVEENPGP	Porcine teschovirus-1
E2A	QCTNYALLKLAGDVESNPGP	Equine rhinitis A virus

Class 2

Consensus Sequence

W-x-x-x-x-E-E-G-I-E-x-x-P-N-P-G-P

Key Features

• N-terminal: Invariant tryptophan
• Central motif: EEGIE (Glu-Glu-Gly-Ile-Glu)
• C-terminal: PNPGP or PHPGP

Representative Peptides

Name	Sequence	Source
F2A	VKQTLNFDLLKLAGDVESNPGP	Foot-and-mouth disease virus

Structural Comparison

flowchart LR
    subgraph Class1[Class 1]
        L1[L-rich N-term] --> G1[GDVE] --> N1[NPGP]
    end

    subgraph Class2[Class 2]
        W2[W N-term] --> E2[EEGIE] --> P2[PNPGP]
    end

Figure 1: Structural comparison of 2A peptide classes

Functional Differences

Feature	Class 1	Class 2
Cleavage efficiency	High	High
Host range	Broad	Broad
Position preference	N-terminal	Variable
Biotechnology use	Common (T2A, P2A)	Less common

Cross-Class Homology

Both classes share:

1. C-terminal GP dipeptide - site of ribosomal skipping
2. Proline at P position - essential for function
3. Upstream asparagine/proline - contributes to efficiency

WarningCross-Matches

HMM searches may identify partial cross-matches between classes due to the conserved C-terminal region. Always verify the full-length sequence context.

Phylogenetic Distribution

• Class 1: Primarily in picornaviruses, insect viruses
• Class 2: Primarily in aphthoviruses

Selection for Biotechnology

Application	Recommended	Reason
Mammalian expression	T2A, P2A	High efficiency, low immunogenicity
Insect cells	E2A	Native to insect viruses
Balanced expression	P2A	Most consistent stoichiometry