Difference between revisions of "Part:BBa K4365006"

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===References===
 
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===Functional Parameters===
 
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<partinfo>BBa_K4365006 parameters</partinfo>
 
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Revision as of 18:34, 11 October 2022

Signal peptide of MPGI from Magnaporthe grisea

MPGI is a protein found in Magnaporthe grisea and belongs to a family of highly secreted proteins called hydrophobins. The signal peptide from the protein led to turboRFP secretion in Saccharomyces cerevisiae.

Collection of fungal signal peptides
ID Source Species Prediction coefficient [1] turboRFP secretion
sp1 BBa_K4365006 MPGI Magnaporthe grisea 0.9562 Yes
sp2 BBa_K4365007 RodA Aspergillus nidulans 0.9553 Yes
sp3 BBa_K4365008 HYPI Aspergillus fumigatus 0.9703 No
sp4 BBa_K4365009 SsgA Metarhizium anisopliae 0.9126 No
sp5 BBa_K4365010 HCF-4 Cladosporium fulvum 0.9126 Yes
sp6 BBa_K4365025 HCF-5 Cladosporium fulvum 0.9234 NA
sp7 BBa_K4365026 CCG-2 Neurospora crassa 0.9566 NA
sp8 BBa_K4365011 HCF-1 Cladosporium fulvum 0.6850 Yes
sp9 BBa_K4365027 HCF-2 Cladosporium fulvum 0.8144 NA
sp10 BBa_K4365012 HCF-3 Cladosporium fulvum 0.7934 No
sp11 BBa_K4365013 SC3 Schizophyllum commune 0.6327 No
sp12 BBa_K4365014 ABH3 Agaricus bisporus 0.9458 Yes
sp13 BBa_K4365028 COH1 Coprinus cinereus 0.8545 NA
sp14 BBa_K4365015 FBH1 Pleurotus ostreatus 0.3367 Yes
sp15 BBa_K4365016 Aa-PRI2 Agrocybe aegerita 0.6356 Yes
sp16 BBa_K4365033 Hyd-Pt1 Pisolithus tinctorius 0.8352 NA
sp17 BBa_K4365017 HFBI Trichoderma reesei 0.9851 Yes
sp18 BBa_K4365018 HFBII Trichoderma reesei 0.6862 Yes
sp19 BBa_K4365019 α-mating factor Saccharomyces cerevisiae 0.9988 Yes
  1. ↑ José Juan Almagro Armenteros et al. (2019) SignalP 5.0 improves signal peptide predictions using deep neural networks Nature Biotechnology, 37, 420-423, doi: 10.1038/s41587-019-0036-z


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology

Secretion signal peptides can be fused to a protein to direct it through the secretory pathway out of the cell [1]. Such secretion of recombinant proteins facilitates the protein purification process and the removal of GMOs [2]. However, commonly used signal sequences like the *S. cerevisiae* alpha-factor prepro-peptide K4365019 and the HSP150, are very long [2] which makes their use in genetic constructs limited. K4365006 as a part of the collection of signal peptides represents a shorter sequence that could be inserted into the gene of interest simply by PCR and could display higher secretion efficiency.

The sequences of the hydrophobic signal peptide was collected from literature [3] and was extracted via analysis of their sequence using the SignalP - 5.0 signal peptide predictor tool [4].

Hydrophobins

Hydrophobins are a group of small (~100 amino acids) proteins that are expressed in ascomycetes and basidiomycetes, which are very efficiently secreted [2].

After secretion from the fungus, they can assemble on the cell walls to form a hydrophobic coating. For this reason, the functions of hydrophobins are related to cell surface activity. For example, they can cover hyphae so the surface tension can be lowered and the hyphae can breach the water/air interface, thus escaping from the aqueous environment and growing into the aerial hyphae [5]. They also mediate the adhesion of the hyphae to hydrophobic surfaces such as in the case of pathogen-host interaction or symbiosis, as observed in lichens and in ectomycorrhizae (the symbiotic relationship between a fungal symbiont and the roots of a plant) [5]. Hydrophobins also form a hydrophobic sheath to protect the surface of conidia, spores, and caps of fungi against wetting and desiccation [5].

Signal peptide screening

For this reason, we have selected signal peptides originating from a group of highly secreted proteins called hydrophobins. We have then cloned their sequences together with turboRFP into the level 1 shuttle plasmid that we developed, so that they could be expressed after the induction of the inducible *FIG1* promoter. We have then carried out multiple secretion assays, in flasks and in multi-well plates to identify the best ones.

Figure 1: fusion protein used for the secretion assays composed of a signal peptide, a flexible linker, and the turboRFP fluorescent protein. Created with Biorender.com.


Measurement protocols:


References

  1. ↑ Alberts, B. (2015) Molecular Biology of the Cell. 6th Edition, Garland Science, Taylor and Francis Group, New York.
  2. ↑ 2.0 2.1 2.2 Kirsten Kottmeier, Kai Ostermann, Thomas Bley, Gerhard Rödel (2011) Hydrophobin signal sequence mediates efficient secretion of recombinant proteins in Pichia pastoris, Appl Microbiol Biotechnol 91, pages133–141, https://doi.org/10.1007/s00253-011-3246-y
  3. ↑ N J Talbot, D J Ebbole, J E Hamer (1993) Identification and characterization of MPG1, a gene involved in pathogenicity from the rice blast fungus Magnaporthe grisea, The Plant Cell, Volume 5, Issue 11, Pages 1575–1590, https://doi.org/10.1105/tpc.5.11.1575
  4. ↑ José Juan Almagro Armenteros et al. (2019) SignalP 5.0 improves signal peptide predictions using deep neural networks Nature Biotechnology, 37, 420-423, doi: 10.1038/s41587-019-0036-z
  5. ↑ 5.0 5.1 5.2 Khalesi, M., Gebruers, K. & Derdelinckx, G. (2015) Recent Advances in Fungal Hydrophobin Towards Using in Industry, Protein J,  34, 243–255 . https://doi.org/10.1007/s10930-015-9621-2