Part:BBa_K1989050

Triple spytag with eforRed and His-tag, FlAsH-tag tag and signal peptide PhoA

Usage and Biology

Biological material

In the last few years, hydrogens made from natural or synthetic polymers have been investigated due to their extensive application in clinical medicine and synthetic biology. Comparing to traditional biological material, protein-based multifunctional biological material is low-cost, facile and eco-friendly. However, strategies for assembling 3D molecular networks synthesized only by protein molecular remain underdeveloped. The reason why investigating this technology is still tough is lack of protein-based cross linking agents.

Spytag and eforRed

Inspiring from the self-catalysis of isopeptide bond between Lys and Asp in Streptococcus pyogenes fibronectin-binding protein FbaB, researchers split the catalytic domain and obtain two peptide called Spytag(the short one) and Spycatcher(the long one) which are able to form isopeptide bond with the other without any assistant. By fusing Spytag and Spycatcher with functional domains respectively, researchers solve the problem tactfully. In order to using Spytag and Spycatcher system as scaffold, we fused three Spytag spaced by (VPGVG)4 with 6xHistag in N-terminal another functional protein called eforRed in C-terminal. This functional protein is obtained from BBa_K592012 (https://parts.igem.org/Part:BBa_K592012").

Signal peptide

Whether in E. coli or Bacillus Subtilis, the secreted proteins usually contain signal peptides that are essential for export from the cytoplasm. Signal peptides are at the N terminus of target proteins and can be cleaved after secretion. Both E. coli and Bacillus subtilis rely on Sec or Tat machinery to traverse the inner membrane. Detailed description is shown in Figure.1, where three distinct ways are depicted.

As several attributes of our target proteins, such as 3A-SUP, 3B, 3A-mSA, etc. remained unknown, especially their folding state in cells, we were unable to rationally design or choose a signal peptide for each. Nevertheless, we could build a Signal Peptide Repertory to screen for the best SP candidate. Our ultimate goal was to select the most appropriate ones from a huge amount of signal peptides, but owing to the limited time, we had just chosen a limited number of signal peptides to test for its potential to secrete our target proteins. Based on previous studies on their performance of secretion, PhoA was selected in this part.

His-tag

His-tag is an amino acid motif in proteins that contains at least six histidine residues and is widely used for recombinant protein purification because of its affinity for bivalent nickel or cobalt ions.

FlAsH-tag

FlAsH-tag is an alias for a tetracysteine-motif-tag (-FLNCCPGCCMEP-) which binds with high affinity and specificity to a biarsenical dye FlAsH-EDT2 and forms a fluorescent compounds.

Based on our results, the fused protein Histag-PhoA-3A-eforRed-FlAshtag possess both isopeptide bond forming function, coloration ability and secretion . Thus, using Histag-PhoA-3A-eforRed-FlAshtag as a part of hydrogel formation, we could obtain visible hydrogel.

Cultivation, Purification and SDS-PAGE

Cultivation

The part was assembled with T7 promoter and RBS in pET28a plasmid vector. E. coli strain BL21(DE3) harboring the appropriate plasmid was grown at 37 °C in 2xYT medium overnight with suitable concentration of antibiotic. The culture was diluted 100 fold into fresh medium with antibiotic and grown at 37°C to an optical density of 0.6~0.8 at 600 nm, the protein expression was induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cells were grown overnight at 25°C.

Purification

Cells were centrifuged at 8000rpm for 15min at 4°C. Resuspend the cell paste expressing recombinant protein in binding buffer (20 mM Tris-HCl, 0.5 M NaCl, 20 mM imidazole, 1mM β-mercaptoethanol, pH7.4), containing SIGMAFAST™ Protease Inhibitor Cocktail Tablets (SIGMA-ALORICH). Disrupt the cells with sonication for 20 min with suitable power on ice and centrifuge at 18000 rpm for 40 min at 4°C. Remove remaining particles by passing the supernatant through a 0.22 μm filter. The HisTrap™ column (GE Healthcare, Inc.) was equilibrated with binding buffer. Load the sample and wash the column with binding buffer.

Elute the target protein with a linear gradient starting with binding buffer and ending with the same buffer including 500mM imidazole. The eluted fraction containing the target protein were concentrated by Amicon® Ultra Centrifugal Filters (Merck) with a 10 kDa cutoff, then frozen by liquid nitrogen and stored at -80°C.

SDS-PAGE

Protein purification was checked by SDS-PAGE and the resulting protein is quantified by Braford analysis.

References

1. Low KO, Mahadi NM, Illias RM. Optimisation of signal peptide for recombinant protein secretion in bacterial hosts. Appl Microbiol Biotechnol.(2013)97(9):3811–3826.

2. Tjalsma H, Bolhuis A, Jongbloed JD, Bron S, van Dijl JM. Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey

3. Hengen, P.N., Purification of His-Tag fusion proteins from Escherichia coli. Trends in Biochemical Sciences, (1995)20(7): p. 285-286.

4. Haitjema, C.H., et al., Universal Genetic Assay for Engineering Extracellular Protein Expression. ACS Synthetic Biology(2014) 3(2): p. 74-82.

5. Sun, F. et al. Proc. Natl Acad. Sci. USA 111, 11269-11274 (2014).

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]