This is an improvement to the BBa_K1789001 part submitted by iGEM15_NUDT_CHINA. A HisTag and GSG linker were added to the N-terminus of the enzyme indole-3-acetamide hydrolase, and a SpyTag was added to the C-terminus.
Usage and Biology
Indole-3-acetamide hydrolase (IaaH) is an enzyme involved in the biosynthesis of indole-3-aectic acid. IaaH originating from Alcaligenes sp. strain HPC127114 was fused with a SpyTag and a hexahistidine tag (HisTag)1.
The auxin indole-3-acetic acid, is a plant hormone involved in the regulation of plant growth and development2. Indole-3-aecetic acid can be synthesised via the indole-3-acetamide pathway, which converts tryptophan to indole-3-aectic acid in a two-step enzymatic pathway3 (Figure 1). The flavoprotein tryptophan 2-monooxygenase (IaaM) catalyses the oxidative decarboxylation of tryptophan to indole-3-acetamide in the first, rate limiting step of the pathway4. Subsequently, the enzyme indole-3-acetamide hydrolase (IaaH) converts indole-3-acetamide to indole-3-aectic acid1.
Figure 1: The indole-3-acetamide pathway for indole-3-aecetic acid biosynthesis.
The SpyTag forms one component of the SpyTag/SpyCatcher system, which enables covalent attachment of two proteins6. The SpyTag and SpyCatcher system was created by cleaving the CnaB2 domain of the fibronectin-binding protein FbaB derived from Streptococcus pyogenes to form a thirteen residue SpyTag peptide and a 116-residue SpyCatcher peptide6. The SpyTag (1.1 kDa) and SpyCatcher (12 kDa) form an irreversible intramolecular isopeptide bond between Asp117 on SpyTag and Lys31 on SpyCatcher6, spontaneously and specifically binding to each other so that they can be used as attachment mechanisms to create new, self-assembling protein arrangements6 (Figure 2).
It is particularly useful because neither component needs to be at the C or N terminus7, and the effect on the attached protein’s activity appears to be negligible10. It also reported as useful in a variety of reaction conditions, with Howarth showing that the SpyTag/SpyCatcher “had a high yield...required only simple mixing (and) tolerated diverse conditions (pH, buffer components and temperature)”8.
Figure 2: A spontaneous isopeptide bond forms between SpyTag and SpyCatcher. Image created using PDB ID: 4MLS9
A HisTag (six consecutive histidine residues, also known as a hexahistidine tag) was added to IaaH to enable purification, utilising the affinity of the HisTag for nickel ions for Immobilised Metal Affinity Chromatography purification5.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 1128
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 661
- 1000COMPATIBLE WITH RFC
IaaH (BBa_K1789001) and His IaaH-SpyTag (BBa_K2710005) were subcloned into the multiple cloning site of pET19b for expression in E. coli T7 Express (NEB) and purified by Immobilised Metal Affinity Chromatography (IMAC). The purification was analysed by SDS-PAGE (Figure 3). The IaaH enzyme encoded by BBa_K1789001 was not His-tagged and thus unable to be purified by IMAC as seen by the absence of a distinct band at the expected molecular weight range (49 kDa). In contrast, the improved His-IaaH-SpyTag was successfully purified as reflected by the clear bands seen at the expected molecular weight range (53 kDa) in the elution lanes.
Figure 3: SDS-PAGE analysis of IMAC purification of IaaH (BBa_K1789001) and His-IaaH-SpyTag (BBa_K2710005). SeeBlue Plus 2 Pre-stained Protein Standard (Invitrogen) was used as the molecular weight standard. Lanes are labelled as flow through (FT), wash (W) and elutions (E1 and E2). Unsuccessful purification of IaaH without His-tag (BBa_K1789001) (MW: 49 kDa) (left) and successful purification of His IaaH-SpyT (MW: 53 kDa) (right).
HisTagged IaaH fused with SpyTag and proteins fused to SpyCatcher (aPFD-SpyCatcher, gPFD-SpyCatcher and SpyCatcher-gPFD-SpyCatcher) were mixed at a concentration of 3 µM and 15 µM respectively in a total volume of 250 µL in PBS pH 8, and incubated at room temperature. After 0, 10, 20 and 30 minutes of incubation, a 10 µL sample was taken and boiled with 5 µL of 4x Bolt LDS sample buffer for 10 minutes at 95°C to cease SpyCatcher reactivity while preserving any covalent interactions. The samples were then examined on SDS-PAGE.
A higher molecular weight band, consistent with a fusion of aPFD-SpyC and IaaH-SpyT (83 kDa), emerges after 10 minutes of reaction and increases in intensity as reaction time increases. In addition, the disappearance of aPFD-SpyC band as reaction time increases suggests that a high proportion of aPFD-SpyC has reacted with the SpyTag on the enzyme.
Figure 4: aPFD-SpyC covalently attaches to IaaH-SpyT. The bands indicating successful attachment of IaaH-SpyT to aPFD-SpyC are boxed in red.
Successful attachment of His IaaH-SpyTag to gPFD-SpyC and gPFD with an N- and C-terminal SpyCatcher fusion (SpyC-gPFD-SpyC) was also demonstrated by SDS-PAGE (Figure 5). A single higher molecular weight band for IaaH-SpyT/gPFD-SpyC reaction emerges over the time course of the experiment, whereas two higher molecular weight bands emerge for the IaaH-SpyT/SpyC-gPFD-SpyC reaction.
Figure 5: SDS-PAGE of His IaaH-SpyTag with gPFD-SpyC and SpyC-gPFD-SpyC. gPFD-SpyC and SpyC-gPFD-SpyC covalently attaches to IaaH-SpyT. The bands indicating successful attachment of IaaH-SpyT to gPFD-SpyC are boxed in red. Bands indicating successful attachment of IaaH-SpyT to SpyC-gPFD-SpyC are boxed in pink.
Wild-type gPFD polymerises to form filamentous structures. Transmission Electron Microscopy (TEM) was performed on gPFD-SpyC attached to IaaH-SpyT to investigate the effect of the attachment of the enzyme on filament formation. Attachment via the SpyTag/SpyCatcher mechanism appears to distort the filaments (Figure 6).
Figure 6: TEM demonstrates that gPFD and gPFD mutants are able to assemble and form filaments. A: Wild-type gPFD filaments. B: gPFD-SpyC filaments, indicating that gPFD is able to form filaments when fused to a SpyCatcher. C: gPFD-SpyC reacted with IaaH-SpyT. Clumps of filaments, or curled filaments were observed.
- Mishra, P., Kaur, S., Sharma, A. N. & Jolly, R. S. Characterization of an Indole-3-Acetamide Hydrolase from Alcaligenes faecalis subsp. parafaecalis and Its Application in Efficient Preparation of Both Enantiomers of Chiral Building Block 2,3-Dihydro-1,4-Benzodioxin-2-Carboxylic Acid. PLoS One 11, e0159009 (2016).
- Davies, P. J. Plant Hormones: Biosynthesis, Signal Transduction, Action! , (Springer Netherlands, 2007).
- Spaepen, S., Vanderleyden, J. & Remans, R. Indole-3-acetic acid in microbial and microorganism-plant signaling. FEMS Microbiol Rev 31, 425-448, doi:10.1111/j.1574-6976.2007.00072.x (2007).
- Gaweska, H. M., Taylor, A. B., Hart, P. J. & Fitzpatrick, P. F. Structure of the flavoprotein tryptophan 2-monooxygenase, a key enzyme in the formation of galls in plants. Biochemistry 52, 2620–6 (2013).
- Hochuli, E., Dobeli, H. & Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr 411, 177-184 (1987).
- Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proc Natl Acad Sci U S A 109, E690-697, doi:10.1073/pnas.1115485109 (2012).
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- Reddington, S. C. & Howarth, M. Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher. Curr Opin Chem Biol 29, 94-99, doi:10.1016/j.cbpa.2015.10.002 (2015).
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