Tag

Part:BBa_K4365010

Designed by: Svetlana Iarovenko   Group: iGEM22_TU_Dresden   (2022-09-29)

Signal peptide of HCF-4 from Cladosporium fulvum

GATA4 is a member of the GATA family of zinc finger transcription factor, which regulates gene transcription by binding to GATA elements. GATA4 was originally discovered as a regulator of cardiac development and subsequently identified as a major regulator of adult cardiac hypertrophy. GATA4 is expressed in both embryo and adult cardiomyocytes where it functions as a transcriptional regulator for many cardiac genes, and also regulates hypertrophic growth of the heart. GATA4 promotes cardiac morphogenesis, cardiomyocytes survival, and maintains cardiac function in the adult heart. We aim to use GATA 4 transcription factor to synthesize a fusion protein to enter the cell by endocytosis, in order to suppress the activation of Hepatic stellate cell. Thus, the symptoms consequential with primary chronic liver disease is suppressed by deactivating active HSCs. <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K4365019">K4365019</a></html>.

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][3] which makes their use in genetic constructs limited. K4365010 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

Signal peptide siquence was cloned together with turboRFP K4365020 into the level 1 shuttle plasmid K4365022 (Figure 1).

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.

After the transformation with the shuttle plasmid, the yeast colonies were grown on W0 medium plates supplemented with histidine, leucine, and methionine and were used for secretion assays in flasks and in a 48-well plate. The α-mating factor pre-pro signal peptide K4365019 was used as a positive control as it has been shown to be able to secrete proteins [8]. A turboRFP without signal peptide was employed as a negative control to account for the amount of free turboRFP present in the media due to cell lysis.

Secretion measurement protocols:

The first secretion experiment was conducted in flasks and showed (Figure 2A) that cell lysis was low as indicated by the low fluorescence of the negative control. In order to have consistent results and ensure confidence we set up biological triplicates. For this, turboRFP secretion was measured using 48-well plate approach. Cells with induced expression were pipetted into the wells of the plate and grown overnight. Starting at 1 hour after induction, samples were taken and the growth rate was monitored by measuring turbidity (NTU) and the development of fluorescence in the supernatant was measured on a plate reader.

With both approaches K4365010 exhibited increased turboRFP secretion in comparison to positive control (Figure 2).

Figure 2: A secretion by signal peptides in flasks. B secretion assay in 48-well plate with an initial cell density of 1 OD. The canonical α-mating factor pre-pro signal peptide [8] (dark grey + control) was used as a positive control as it has been shown to be able to secrete proteins. A turboRFP without signal peptide was employed as a negative control to account for cell lysis (light grey, - control). The signal corresponding to K4365010 is depicted as a dashed line. Error bars represent mean ± SD.

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. ↑ 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.
  4. ↑ G. C. Segers, W. Hamada, R. P. Oliver, P. D. Spanu (1999) Isolation and characterisation of five different hydrophobin-encoding cDNAs from the fungal tomato pathogen Cladosporium fulvum, Molecular and General Genetics MGG volume 261, pages644–652 https://doi.org/10.1007/s004380050007
  5. ↑ 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
  6. ↑ 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. ↑ 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
  8. ↑ 8.0 8.1 Peñas M. M. et al. (1998) Identification, characterization, and In situ detection of a fruit-body-specific hydrophobin of Pleurotus ostreatus, Appl Environ Microbiol. 64(10):4028-34. doi: 10.1128/AEM.64.10.4028-4034.1998. PMID: 9758836; PMCID: PMC106595.
[edit]
Categories
//proteindomain/localization
Parameters
None