2A peptides

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Fusion protein (top) and cleavage of a fusion protein with 2A peptide (bottom)

2A peptides are a class of viral structural motifs that occur in proteins and share them during translation .

properties

The first 2A peptide sequence was the foot-and-mouth disease virus (as translation of English . Foot-and-mouth disease virus , abbreviated FMDV) discovered. 2A peptides have been described in various mammalian viruses (the picornavirus family ) and many insect viruses. It was named after the name of the gene of a peptidase called 2A pro of the virus in which it was first found. So the P2A peptide derived from Porcine teschovirus 1 (as a translation of English . Porcine teschovirus-1 , abbreviated PTV1). Here, the 2A sequence results in the co-translational division of two proteins within a single open reading frame (as a translation of English . Open reading frame , abbreviated ORF). It is assumed that the 2A sequence inhibits the formation of a regular peptide bond on the ribosome and that the peptides are consequently separated during translation . Presumably, the formation of a Gly - Pro bond C -terminal on the 2A peptide is skipped during translation on the ribosome . The exact mechanism is still unknown. The cleavage always takes place before the C -terminal proline of the 2A peptide. Therefore, the second ( C -terminal) peptide receives a proline as the N -terminus and the first ( N -terminal) peptide receives the remaining 2A sequence as the C -terminus.

application

2A peptides are used genetically in vector design to split a protein into two proteins at a specific location after expression . This is known as polycistronic expression. Their advantage over the analogously used IRES sequences is the equimolar expression of both proteins from a common promoter , while with IRES the second protein is formed in smaller quantities. A gene expression of up to five proteins with four intervening 2A peptides from one gene has been described. Of various 2A peptides examined, P2A was the 2A peptide with the most efficient cleavage, while F2A was the one with the least efficiency. In F2A, up to 50% of the fusion proteins can remain uncleaved, which can result in unintended new functions. 2A peptides can be used in addition to mammalian and insect cells in Saccharomyces cerevisiae , coccidia and (with modifications) in plants . The C -terminal protein should not have myristoylation , since 2A peptides can then change the cellular localization. The separation of the proteins can be checked by transfecting a vector which codes for a protein 2A peptide protein in cell cultures with subsequent Western blotting . Since short peptide sequences remain at the termini of the separated proteins after the cleavage of the 2A peptides, this can lead to functional disorders of these proteins.

Representative

The following table lists the representatives of the 2A peptides that are most relevant in genetic engineering. A "GSG" linker ( Gly - Ser -Gly) N -terminal in front of the 2A peptide can also increase the effectiveness. 2A peptides have the consensus sequence DxExNPGP .

designation sequence
T2A EGRGSLLTCGDVEENPG'P
P2A ATNFSLLKQAGDVEENPG'P
E2A QCTNYALLKLAGDVESNPG'P
F2A VKQTLNFDLLKLAGDVESNPG'P

Individual evidence

  1. ↑ a b c d e f A. L. Szymczak-Workman, KM Vignali, DAA Vignali: Design and Construction of 2A Peptide-Linked Multicistronic Vectors . In: Cold Spring Harbor Protocols . 2012, No. 2, 2012, ISSN  1559-6095 , pp. 199-204. doi : 10.1101 / pdb.ip067876 . PMID 22301656 .
  2. ↑ a b c G. A. Luke, P. de Felipe, A. Lukashev, SE Kallioinen, EA Bruno, MD Ryan: Occurrence, function and evolutionary origins of '2A-like' sequences in virus genomes. In: The Journal of general virology. Volume 89, Pt 4April 2008, pp. 1036-1042, doi : 10.1099 / vir.0.83428-0 , PMID 18343847 , PMC 2885027 (free full text).
  3. ^ MD Ryan, AM King, GP Thomas: Cleavage of foot-and-mouth disease virus polyprotein is mediated by residues located within a 19 amino acid sequence. In: The Journal of general virology. Volume 72 (Pt 11), November 1991, pp. 2727-2732, doi : 10.1099 / 0022-1317-72-11-2727 , PMID 1658199 .
  4. ↑ ML Donnelly, G. Luke, A. Mehrotra, X. Li, LE Hughes, D. Gani, MD Ryan: Analysis of the aphthovirus 2A / 2B polyprotein 'cleavage' mechanism indicates not a proteolytic reaction, but a novel translational effect: a putative ribosomal 'skip'. In: The Journal of general virology. Volume 82, Pt 5 May 2001, pp. 1013-1025, doi : 10.1099 / 0022-1317-82-5-1013 , PMID 11297676 .
  5. ↑ P. Sharma, F. Yan, VA Doronina, H. Escuin-Ordinas, MD Ryan, JD Brown: 2A peptides provide distinct solutions to driving stop-carry on translational recoding. In: Nucleic acids research. Volume 40, number 7, April 2012, pp. 3143-3151, doi : 10.1093 / nar / gkr1176 , PMID 22140113 , PMC 3326317 (free full text).
  6. ↑ Y. Wang, F. Wang, R. Wang, P. Zhao, Q. Xia: 2A self-cleaving peptide-based multi-gene expression system in the silkworm Bombyx mori. In: Scientific Reports . Volume 5, November 2015, p. 16273, doi : 10.1038 / srep16273 , PMID 26537835 , PMC 4633692 (free full text).
  7. ^ AL Szymczak, DA Vignali: Development of 2A peptide-based strategies in the design of multicistronic vectors. In: Expert Opinion on Biological Therapy. Volume 5, Number 5, May 2005, pp. 627-638, doi : 10.1517 / 14712598.5.5.627 , PMID 15934839 .
  8. ↑ a b c d Ziqing Liu, Olivia Chen, J. Blake Joseph Wall, Michael Zheng, Yang Zhou, Li Wang, Haley Ruth Vaseghi, Li Qian, Jiandong Liu: Systematic comparison of 2A peptides for cloning multi-genes in a polycistronic vector . In: Scientific Reports . 7, No. 1, 2017, ISSN  2045-2322 , p. 2193. bibcode : 2017NatSR ... 7.2193L . doi : 10.1038 / s41598-017-02460-2 . PMID 28526819 . PMC 5438344 (free full text).
  9. ↑ a b G.A. Luke, MD Ryan: "Therapeutic applications of the 'NPGP' family of viral 2As". In: Reviews in Medical Virology. Volume 28, number 6, 11 2018, p. E2001, doi : 10.1002 / rmv.2001 , PMID 30094875 .
  10. ↑ JH Kim, SR Lee, LH Li, HJ Park, JH Park, KY Lee, MK Kim, BA Shin, SY Choi: High cleavage efficiency of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. In: PLOS ONE . Volume 6, number 4, 2011, p. E18556, doi : 10.1371 / journal.pone.0018556 , PMID 21602908 , PMC 3084703 (free full text).
  11. ↑ S. Velychko, K. Kang, SM Kim, TH Kwak, KP Kim, C. Park, K. Hong, C. Chung, JK Hyun, CM MacCarthy, G. Wu, HR Schöler, DW Han: Fusion of Reprogramming Factors Alter the Trajectory of Somatic Lineage Conversion. In: Cell Reports. Volume 27, number 1, 04 2019, pp. 30-39.e4, doi : 10.1016 / j.celrep.2019.03.023 , PMID 30943410 .
  12. ↑ TM Souza-Moreira, C. Navarrete, X. Chen, CF Zanelli, SR Valentini, M. Furlan, J. Nielsen, A. Krivoruchko: Screening of 2A peptides for polycistronic gene expression in yeast. In: FEMS yeast research. Volume 18, number 5, 08 2018, p., Doi : 10.1093 / femsyr / foy036 , PMID 29617770 .
  13. ↑ X. Tang, X. Liu, G. Tao, M. Qin, G. Yin, J. Suo, X. Suo: "Self-cleaving" 2A peptide from porcine teschovirus-1 mediates cleavage of dual fluorescent proteins in transgenic Eimeria tenella. In: Veterinary research. Volume 47, number 1, 06 2016, p. 68, doi : 10.1186 / s13567-016-0351-z , PMID 27352927 , PMC 4924277 (free full text).
  14. ↑ S. Burén, C. Ortega-Villasante, K. Otvös, G. Samuelsson, L. Bakó, A. Villarejo: Use of the foot-and-mouth disease virus 2A peptide co-expression system to study intracellular protein trafficking in Arabidopsis . In: PLOS ONE . Volume 7, number 12, 2012, p. E51973, doi : 10.1371 / journal.pone.0051973 , PMID 23251667 , PMC 3522588 (free full text).
  15. ↑ B. Zhang, M. Rapolu, S. Kumar, M. Gupta, Z. Liang, Z. Han, P. Williams, WW Su: Coordinated protein co-expression in plants by harnessing the synergy between an intein and a viral 2A peptides. In: Plant biotechnology journal. Volume 15, number 6, June 2017, pp. 718–728, doi : 10.1111 / pbi.12670 , PMID 27879048 , PMC 5425387 (free full text).
  16. ↑ S. Hadpech, W. Jinathep, p Saoin, W. Thongkum, K. Chupradit, U. Yasamut, p Moonmuang, C. Tayapiwatana: Impairment of a membrane-targeting protein translated from a gene downstream of a "self- cleaving "T2A peptide conjunction. In: Protein expression and purification. Volume 150, 10 2018, pp. 17-25, doi : 10.1016 / j.pep.2018.05.002 , PMID 29733907 .
  17. ↑ AL Szymczak-Workman, KM Vignali, DA Vignali: Verification of 2A peptide cleavage. In: Cold Spring Harbor protocols. Volume 2012, number 2, February 2012, pp. 255-257, doi : 10.1101 / pdb.prot067892 , PMID 22301658 .
  18. ↑ X. Yang, A. Cheng, M. Wang, R. Jia, K. Sun, K. Pan, Q. Yang, Y. Wu, D. Zhu, S. Chen, M. Liu, XX Zhao, X. Chen: Structures and Corresponding Functions of Five Types of Picornaviral 2A Protein. In: Frontiers in Microbiology. Volume 8, 2017, p. 1373, doi : 10.3389 / fmicb.2017.01373 , PMID 28785248 , PMC 5519566 (free full text).