Using a viral 2A peptide-based strategy to reconstruct the bovine P450scc steroidogenic system in S. cerevisiae

Efimova, Vera S.; Isaeva, Ludmila V.; Orekhov, Philipp; Bozdaganyan, Marine E.; Rubtsov, Mikhail A.; Novikova, Ludmila A.

doi:10.1016/j.jbiotec.2020.10.028

Cited by 6 publications

(4 citation statements)

References 68 publications

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“…Our data are consistent with the reports that the highest protein expression was found at the first position in tri-cistronic constructs (Liu et al, 2017). It is also found in co-expression of cytochrome P450cc, adrenodoxin and adrenodoxin linked by two 2A peptides, the cleavage efficiency of the first 2A ranged 90-96%, while the cleavage efficiency of the second 2A peptides ranged from 11 to 68% (Efimova et al, 2021).…”

Section: Co-expression Of Paenibacillus Polymyxa Nifh and Klebsiella ...supporting

confidence: 92%

Assembly of nitrogenase biosynthetic pathway in Saccharomyces cerevisiae by using polyprotein strategy

Wang

Shang²,

Liu³

et al. 2023

Front. Microbiol.

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Nitrogenase in some bacteria and archaea catalyzes conversion of N2 to ammonia. To reconstitute a nitrogenase biosynthetic pathway in a eukaryotic host is still a challenge, since synthesis of nitrogenase requires a large number of nif (nitrogen fixation) genes. Viral 2A peptide mediated “cleavage” of polyprotein is one of strategies for multigene co-expression. Here, we show that cleavage efficiency of NifB-2A-NifH polyprotein linked by four different 2A peptides (P2A, T2A, E2A, and F2A) in Saccharomyces cerevisiae ranges from ~50% to ~90%. The presence of a 2A tail in NifB, NifH, and NifD does not affect their activity. Western blotting shows that 9 Nif proteins (NifB, NifH, NifD, NifK, NifE, NifN, NifX, HesA, and NifV) from Paenibacillus polymyxa that are fused into two polyproteins via 2A peptides are co-expressed in S. cerevisiae. Expressed NifH from Klebsiella oxytoca NifU and NifS and P. polymyxa NifH fusion linked via 2A in S. cerevisiae exhibits Fe protein activity.

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Section: Co-expression Of Paenibacillus Polymyxa Nifh and Klebsiella ...supporting

confidence: 92%

Assembly of nitrogenase biosynthetic pathway in Saccharomyces cerevisiae by using polyprotein strategy

Wang

Shang²,

Liu³

et al. 2023

Front. Microbiol.

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“…Herein pig IL-2 and FBC genes were firstly co-expressed using the self-cleavage 2A peptide technique, which permits multiple genes to be equally expressed in the same vector [ 26 , 27 ]. Our results showed that recombinant IL-2 and FBC significantly promoted the proliferation of pig lymphoblast in vitro and increased the number of leukocytes, CD4+ and CD8+ T cells, IgG, and the gene expressions of TLRs (TLR1,4,6,9), antimicrobial peptides (CRP4 and CRAMP) and interleukins (IL-2, 4, 6, 7, 12, 15, and 23), IFN-γ and TNF-α in the blood in vivo both before and after challenge.…”

Section: Discussionmentioning

confidence: 99%

Co-Expression of Pig IL-2 and Fusion Bovine Cathelicidin Gene by Recombinant Plasmids in Yeast and Their Promotion of Mouse Antibacterial Defense

Chen

Peng

et al. 2022

Biology

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In order to develop an effective and safe immunomodulator to enhance the antimicrobial bioactivity and immunity of animals against infectious bacterial diseases, a recombinant plasmid pGAPZαA-IL2-B co-expressing pig interleukin-2 (PIL-2) and fused bovine cathelicidin (FBC) genes were constructed using the 2A self-cleavage technique. After being expressed in Pichia pastoris strain SMD1168, the recombinant yeast was administered orally to 5-week-old female ICR mice. The control mice were similarly dosed with P. pastoris with a blank plasmid or FBC recombinant plasmid alone. At 28 days post-treatment, the mice were challenged intraperitoneally with virulent strains of either E. coli or S. aureus. Compared with the control groups, the mice that received recombinant yeast co-expressing PIL-2/FBC manifested significant increases in the number of leukocytes, CD4+ and CD8+ T cells, IgG, and the gene expressions of TLRs(TLR1,4,6,9), antimicrobial peptides(CRP4 and CRAMP) and cytokines (IL-2, 4, 6, 7, 12, 15, 23, IFN-γ, and TNF-α) in the blood. Furthermore, the treated mice displayed significantly higher survival than the other two control groups after the challenge. These results suggest that the antimicrobial activity and immunity of animals can be effectively enhanced by the in vivo co-expression of IL-2 and the FBS gene, which can facilitate the development of new immunopotentiation molecules to overcome the infection of antibiotic-resistant bacteria.

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“…Interestingly, P2A 19 and T2A 18 functions in Drosophila are approximately equivalent both in cultured cells and in vivo [80]-given their poor efficiency at polypeptide separation, E2A and F2A may not be useful in Drosophila [80,81]. A study to characterise the 2A system for metabolic engineering applications in Saccharomyces cerevisiae showed that the F2A 19 , T2A 18 , and P2A 19 sequences are functional in S. cerevisiae cells [82]-earlier analysis in S. cerevisiae indicated that ERBV-1 (Equine rhinitis B virus 1) 2A had the highest cleaving efficiency among 22 viral 2A sequences tested [48]. For potential upcoming amoebabased bioprocesses codon optimised P2A 19 , T2A 18 , E2A 20 and F2A 22 were screened for activity in Dictyostelium discoideum [45].…”

Section: Comparison Of Active 2a Sequencesmentioning

confidence: 99%

The 2A Story: The End of the Beginning

A. Luke,

D. Ryan

2024

Beyond the Blueprint - Decoding the Elegance of Gene Expression [Working Title]

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Translational control of viral gene expression is a fundamental process essential for the vitality of all viruses. In special cases, signals encoded in the mRNA reprogram the ribosome to read the message in a different way, a process termed “translational recoding”. The 2A region of the foot-and-mouth disease virus (FMDV) encodes a short sequence, only 18 amino acids, that mediates self-processing by a novel translational effect “ribosome skipping” rather than proteolysis. Briefly, 2A interacts with the ribosome exit tunnel to inhibit peptide bond formation at the C terminus of the 2A sequence. Translation terminates at this point, but then resumes elongation, creating a second independent protein product. Thus, discrete proteins can be produced from a single transcript. The 2A sequence is particularly useful in vector strategies (AAV and retroviral vectors) where the capacity to incorporate foreign DNA is limited. Use of 2A and “2A-like” peptides to link the sequences encoding several proteins in the same open reading frame has led to their increasing use as important tools in biotechnology and biomedicine. This technology has been crucial for the visual tracking of expressed proteins, human gene therapies targeting cancer, production of induced human pluripotent stem cells for regenerative medicine, creation of transgenic animals and plants and the improvement of CRISPR-Cas9 and TALEN genome editing methods.

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Using a viral 2A peptide-based strategy to reconstruct the bovine P450scc steroidogenic system in S. cerevisiae

Cited by 6 publications

References 68 publications

Assembly of nitrogenase biosynthetic pathway in Saccharomyces cerevisiae by using polyprotein strategy

Assembly of nitrogenase biosynthetic pathway in Saccharomyces cerevisiae by using polyprotein strategy

Co-Expression of Pig IL-2 and Fusion Bovine Cathelicidin Gene by Recombinant Plasmids in Yeast and Their Promotion of Mouse Antibacterial Defense

The 2A Story: The End of the Beginning

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