Difference between revisions of "Part:BBa K2710005"
Line 19: Line 19:
  
 
===Characterisation===
 
===Characterisation===
 +
<p><b> Protein Purification</b></p>
 
<p> IaaH (BBa_K1789001)  and 6xHis IaaH-SpyTag (BBa_K2710005) were subcloned into the multiple cloning site of pET19b for expression in <i>E. coli</i> T7 Express (NEB) and purified by Immobilised Metal Affinity Chromatography (IMAC). The purification was analysed by SDS-PAGE (figure 1). The IaaH enzyme encoded by BBa_K1789001 was not His-tagged and thus unable to be purified by IMAC. The improved 6xHis IaaH-SpyTag was successfully purified as reflected by the clear bands seen at the expected molecular weight range (53 kDa) in the elution lanes. </p>
 
<p> IaaH (BBa_K1789001)  and 6xHis IaaH-SpyTag (BBa_K2710005) were subcloned into the multiple cloning site of pET19b for expression in <i>E. coli</i> T7 Express (NEB) and purified by Immobilised Metal Affinity Chromatography (IMAC). The purification was analysed by SDS-PAGE (figure 1). The IaaH enzyme encoded by BBa_K1789001 was not His-tagged and thus unable to be purified by IMAC. The improved 6xHis IaaH-SpyTag was successfully purified as reflected by the clear bands seen at the expected molecular weight range (53 kDa) in the elution lanes. </p>
  
Line 27: Line 28:
 
<br />
 
<br />
  
 +
<p></b>SpyCatcher/Tag Assembly</b></p>
  
 +
<p>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.</p>
 +
<p>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.</p>
 +
 +
https://static.igem.org/mediawiki/2018/thumb/b/b7/T--UNSW_Australia--assembly3.jpeg/291px-T--UNSW_Australia--assembly3.jpeg
 +
<p class="figure-legend fig2-leg"><b>Figure 2:</b> aPFD-SpyC covalently attaches to IaaH-SpyT. The bands indicating successful attachment of IaaH-SpyT to aPFD-SpyC are boxed in red. </p>
 +
<br />
  
  

Revision as of 07:19, 17 October 2018


His-IaaH-SpyTag

This is an improvement on the BBa_K1789001 part submitted by iGEM15_NUDT_CHINA. A HisTag and SpyTag were added to the enzyme ndole-3-acetamide hydrolase.


Usage and Biology

Indole-3-acetamide hydrolase 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).

The auxin indole-3-acetic acid, is a plant hormone involved in the regulation of plant growth and development1. 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 pathway2. The flavoprotein tryptophan 2-monooxygenase (IaaM) catalyses the oxidative decarboxylation of tryptophan to indole-3-acetamide in the first, rate limiting step of the pathway3. Subsequently, the enzyme indole-3-acetamide hydrolase (IaaH) converts indole-3-acetamide to indole-3-aectic acid4.


The SpyTag forms one component of the SpyTag/SpyCatcher system, which enables covalent attachment of two proteins5. 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 peptide5. The SpyTag (1.1 kDa) and SpyCatcher (12 kDa) form an irreversible intramolecular isopeptide bond between Asp117 on SpyTag and Lys31 on SpyCatcher5, spontaneously and specifically binding to each other so that they can be used as attachment mechanisms to create new, self-assembling protein arrangements6.

It is particularly useful because neither component needs to be at the C or N terminus6, 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)”7.


A 6x 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 purification8.


Characterisation

Protein Purification

IaaH (BBa_K1789001) and 6xHis 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 1). The IaaH enzyme encoded by BBa_K1789001 was not His-tagged and thus unable to be purified by IMAC. The improved 6xHis IaaH-SpyTag was successfully purified as reflected by the clear bands seen at the expected molecular weight range (53 kDa) in the elution lanes.


800px-T--UNSW_Australia--IaaH-purifications.jpeg

Figure 1: SDS-PAGE analysis of IMAC purification of IaaH (BBa_K1789001) and 6xHis IaaH-SpyTag (BBa_K2710005). SeeBlue Plus 2 Pre-stained Protein Standard (Invitrogen) was used as the molecular weight standard. Lanes are labelled as cell lysate (L), flow through (FT), wash (W) and elutions (E1, E2, E3, E4, E5). Unsuccessful purification of IaaH without His-tag( BBa_K1789001) (MW: 49 kDa) (left) and successful purification of 6xHis IaaH-SpyT (MW: 53 kDa) (right)


</b>SpyCatcher/Tag Assembly</b>

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.

291px-T--UNSW_Australia--assembly3.jpeg

Figure 2: aPFD-SpyC covalently attaches to IaaH-SpyT. The bands indicating successful attachment of IaaH-SpyT to aPFD-SpyC are boxed in red.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1128
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 661
  • 1000
    COMPATIBLE WITH RFC[1000]


References

  1. Davies, P. J. Plant Hormones: Biosynthesis, Signal Transduction, Action! , (Springer Netherlands, 2007).
  2. 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).
  3. 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).
  4. 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).
  5. 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).
  6. Domeradzka, N. E., Werten, M. W., Wolf, F. A. & 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).
  7. 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).
  8. 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).


</div>