Difference between revisions of "Part:BBa K2710005"

Revision as of 14:13, 17 October 2018

His-IaaH-SpyTag

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. 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. Indole-3-acetamide hydrolase originating from Alcaligenes sp. strain HPC127114 was fused with a SpyTag and a hexahistidine tag (his tag)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.

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 purification¹¹.

Sequence and Features

Characterisation

Protein Purification

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. 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).

SpyCatcher/Tag Assembly

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 95oC 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 3]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.