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.

Introduction

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 acid5.


Figure 1: The indole-3-acetamide pathway for indole-3-aecetic acid biosynthesis.1.


Improvements

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.


Characterisation

Figure 3: DNA constructs designed by Brian Ee. Images were generated by Benchling.

References

  1. Gibson, D. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods 6, 343-345 (2009).
  2. Siegert, R., Leroux, M., Scheufler, C., Hartl, F. & Moarefi, I. Structure of the Molecular Chaperone Prefoldin. Cell 103, 621-632 (2000).
  3. Zakeri, B. et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences 109, E690-E697 (2012).
  4. Vellanoweth, R. & Rabinowitz, J. The influence of ribosome-binding-site elements on translational efficiency in Bacillus subtilis and Escherichia coli in vivo. Molecular Microbiology 6, 1105-1114 (1992).