Team:UNSW Australia/Improve

Improved Part

Abstract

The UNSW iGEM team designed a new IaaH encoding part for improved functionality of the indole-3-aectamide hydrolase (IaaH) enzyme. Improving on the BBa_K89001 part submitted by the 2015 NUDT_China team, a HisTag and GSG linker were added to the N-terminus of the enzyme, and a SpyTag and GSG linker was added to the C-terminus. Following these additions, the team was able to successfully express and purify the new part utilising the HisTag's affinity for nickel ions to purify the enzyme using IMAC purification. We also demonstrated the ability of IaaH with SpyTag to covalently bind with SpyCatcher proteins through SDS-PAGE. More details can be found on the improved part's registry page at BBa_K2710005. Through these improvements, we have expanded the utility of this part twofold. First, the protein can be easily purified from cell lysates, and secondly, it now includes an irreversible attachment mechanism, robust over a range of conditions.

The Enzyme

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.


Improvements

The original part was improved by two key additions; a SpyTag and a HisTag.

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 negligible8. 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)”9.

Figure 2: A spontaneous isopeptide bond forms between SpyTag and SpyCatcher. Image created using PDB ID: 4MLS10


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.


Experimental Characterisation

The improved part was validated by sequence verification and a diagnostic gel. Following this, the original and improved part were cloned into the expression vector pET19B and successfully expressed in E. coli T7 Express cells (NEB). The HisTag and SpyTag improvements were experimentally characterised through SDS-PAGE analysis of IMAC purifications and attachment to SpyCatcher fusion proteins. Refer to the BBa_K2710005 registry page for details of the experimental characterisation.

Through these improvements, we have expanded the utility of this part twofold. First, the protein can be easily purified from cell lysates, and secondly, it now includes an irreversible attachment mechanism, robust over a range of conditions.

Furthermore, as 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 3).


Figure 3: 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.

References

  1. Mishra, P., Kaur, S., Sharma, A. N. and 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).
  2. Davies, P. J. Plant Hormones: Biosynthesis, Signal Transduction, Action! , (Springer Netherlands, 2007).
  3. Spaepen, S., Vanderleyden, J. and 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).
  4. Gaweska, H. M., Taylor, A. B., Hart, P. J. and 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).
  5. Hochuli, E., Dobeli, H. and Schacher, A. New metal chelate adsorbent selective for proteins and peptides containing neighbouring histidine residues. J Chromatogr. 411 177-184 (1987).
  6. 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).
  7. Domeradzka, N. E., Werten, M. W., Wolf, F. A. and de Vries, R. Protein cross-linking tools for the construction of nanomaterials. Curr Opin Biotechnol. 39 61-67, doi:10.1016/j.copbio.2016.01.003 (2016).
  8. Walper, S. A., Turner, K. B. and Medintz, I. L. Enzymatic bioconjugation of nanoparticles: developing specificity and control. Curr Opin Biotechnol. 34 232-241, doi:10.1016/j.copbio.2015.04.003 (2015).
  9. Reddington, S. C. and 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).
  10. Li, L., Fierer, J. O., Rapoport, T. A. and Howarth, M. Structural Analysis and Optimization of the Covalent Association between SpyCatcher and a Peptide Tag. J. Mol. Biol. 426 309–317 (2014).