In molecular biology, a 2A sequence is a short, self-processing peptide sequence that allows scientists to efficiently produce multiple, distinct proteins from a single genetic message. This biological tool enables the simultaneous expression of several proteins, overcoming traditional challenges in gene expression. It aids in studying complex biological pathways and developing new biotechnological applications.
Understanding 2A Sequences
A 2A sequence is a short peptide, typically 18 to 22 amino acids long, incorporated into a larger protein-coding sequence. It facilitates the production of multiple, separate proteins from a single messenger RNA (mRNA) molecule. These sequences were first identified in viruses, such as the foot-and-mouth disease virus, where they enable the virus to produce individual proteins from one continuous genetic transcript.
The 2A sequence is inserted between the coding regions for the desired proteins. When this combined genetic message is translated by the cell’s machinery, the 2A sequence signals the ribosome to “skip” a peptide bond during protein synthesis. This separates the upstream protein from the downstream protein, even though they were initially encoded as one continuous polyprotein. This allows for the simultaneous production of multiple discrete proteins.
The Mechanism of Self-Cleavage
The mechanism of 2A sequences is termed “ribosomal skipping,” “stop-carry on,” or “StopGo” translation. During protein synthesis, the ribosome moves along the mRNA, adding amino acids to form a polypeptide chain. When the ribosome encounters a 2A sequence, specifically a conserved core motif of DxExNPGP, it briefly pauses.
At this point, between a glycine (G) and a proline (P) residue within the 2A sequence, the ribosome fails to form a peptide bond. The growing polypeptide chain, including the upstream protein and the 2A sequence, is released. The ribosome then continues to translate the downstream sequence, initiating a new polypeptide chain that begins with a proline residue. This results in two separate proteins: the upstream protein carries a small remnant of the 2A sequence at its C-terminus, and the downstream protein begins with an extra proline.
Key Applications in Biotechnology
2A sequences are valuable tools in biotechnology due to their ability to co-express multiple proteins from a single transcript. In gene therapy, 2A sequences can deliver both a therapeutic gene and a reporter gene simultaneously, allowing researchers to track the delivery and expression of the therapeutic gene by observing the reporter protein.
In research, 2A sequences are employed to express multiple components of signaling pathways or protein complexes, ensuring all necessary proteins are produced at comparable levels. This simplifies the study of complex biological interactions. In vaccine development, 2A sequences facilitate the expression of multiple antigens from a single construct, potentially leading to more comprehensive immune responses.
Different Types and Practical Aspects
Several common types of 2A sequences are widely used, each originating from different viruses. These include F2A (from foot-and-mouth disease virus), P2A (from porcine teschovirus-1), T2A (from Thosea asigna virus), and E2A (from equine rhinitis A virus). While all 2A sequences function through ribosomal skipping, their efficiencies can vary.
Practical considerations for using 2A sequences include their efficiency; ribosomal skipping is not always 100% complete, meaning a small percentage of uncleaved fusion protein may remain. For instance, F2A is less efficient than T2A and P2A, with up to 50% of F2A-linked proteins potentially remaining as a fusion. Additionally, the upstream protein retains a small portion of the 2A sequence, and the downstream protein gains an N-terminal proline. These modifications could affect protein function or elicit an immune response, especially with foreign viral peptides.