Part:BBa_K4365006

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

↑ 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]^[3] 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 ^[4] and was extracted via analysis of their sequence using the SignalP - 5.0 signal peptide predictor tool ^[5].

Identified sequence was codon-optimized for S. cerevisiae using the codon optimization tool on GenScript. Then it was synthesized by IDT as a gBlock together with a flexible glycine linker (K4365005) and the turboRFP gene (K4365020) ^[6].

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 ^[7]. 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) ^[7]. Hydrophobins also form a hydrophobic sheath to protect the surface of conidia, spores, and caps of fungi against wetting and desiccation ^[7].

Signal peptide secretion 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

↑ Alberts, B. (2015) Molecular Biology of the Cell. 6th Edition, Garland Science, Taylor and Francis Group, New York.
↑ ^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
↑ Russo P, Kalkkinen N, Sareneva H, Paakkola J, Makarow M. A heat shock gene from Saccharomyces cerevisiae encoding a secretory glycoprotein. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3671-5. doi: 10.1073/pnas.89.9.3671. Erratum in: Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8857. PMID: 1570286; PMCID: PMC525552.
↑ 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
↑ 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
↑ Merzlyak, E., Goedhart, J., Shcherbo, D. et al. (2007) Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods 4, 555–557 (2007). https://doi.org/10.1038/nmeth1062
↑ ^7.0 ^7.1 ^7.2 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

[signalp-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

[2albert-2] Alberts, B. (2015) Molecular Biology of the Cell. 6th Edition, Garland Science, Taylor and Francis Group, New York.

[1kirsten-3] 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

[4] Russo P, Kalkkinen N, Sareneva H, Paakkola J, Makarow M. A heat shock gene from Saccharomyces cerevisiae encoding a secretory glycoprotein. Proc Natl Acad Sci U S A. 1992 May 1;89(9):3671-5. doi: 10.1073/pnas.89.9.3671. Erratum in: Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8857. PMID: 1570286; PMCID: PMC525552.

[5] 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

[6] 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

[7] Merzlyak, E., Goedhart, J., Shcherbo, D. et al. (2007) Bright monomeric red fluorescent protein with an extended fluorescence lifetime. Nat Methods 4, 555–557 (2007). https://doi.org/10.1038/nmeth1062

[5khal-8] 7.0 ^7.1 ^7.2 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

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