Composite
Part:BBa_K3794005
Designed by: Harshraj Bumia Group: iGEM21_KCL_UK (2021-09-30)
Revision as of 16:57, 21 October 2021 by Harshbumia (Talk | contribs)
ChABC Composite
This composite part acts as a translational unit for the expression of ChABC in E.coli. The LacI regulatory system (BBa_R0010) which encodes the lac promoter and operator allow for IPTG-induced expression in E.coli. BBa_B0010, a T1 terminator from E.coli rrnB also allows for termination of transcription. As the ChABC basic part encodes a 6xHis tag and TEV cleavage site, protein purification using a Ni-NTA column can be achieved after expression of ChABC using this composite part.
Expression of composite part in E.coli produces a thermostabilised version of the ChABC enzyme derived from (PDB 1HN0), with an N-Terminal 6xHis tag and a TEV cleavage site.
This composite part can be utilised with any RFC[10] compatible plasmid backbone.
This part, BBa_K3794005, was directly synthesised by IDT.
Usage and Biology
Introduction
ChABC is a bacterial enzyme that digests Chondroitin Sulphate Proteoglycans (CSPGs) to encourage plasticity and axonal regeneration in the Central Nervous System (CNS). The enzyme has been studied in various CNS injuries including Spinal Cord Injuries (SCI) in vitro and in vivo to overcome the growth inhibitory environment posed by CSPGs which are elevated in the extracellular matrix of the injury site. Studies investigating the therapeutic potential of ChABC in treating SCI have demonstrated that ChABC promotes axonal spouting and aids in functional recovery. Studies have shown that ChABC works by cleaving glycosaminoglycan (GAG) chains of CSPGs, releasing growth-promoting and neurotrophic factors bound to the GAG chains, which then promote axonal outgrowth. ChABC also induces microglial cells and macrophages to take on a neuroprotective and pro-inflammatory role. ChABC administered into the lesion site, by initiating a reparatory process that allows axonal sprouting and functional recovery, has promising potential to be utilised in clinical treatment for SCI.Modelling
DNA Amplification and DNA Purification
Due to the large size of this composite part, 3322bp, it was synthesised in two samples. PCR using a thermal cycler was used for the amplification of both samples of this composite part. PCR products of ChABC and a control sample were visualised using a 1% Agarose gel electrophoresis. ChABC is 3322bp long, and distinct strong bands can be seen in Lane 3 and Land 4 which correlate to the ~3000bp region of the Promega 100bp DNA Ladder, thereby confirming PCR of ChABC was successful.
Figure 2: SnapGene simulation of 1% Agarose gel electrophoresis. Lane 1: Promega 1kb DNA Ladder. Lane 2: -- Lane 3: ChABC Sample 1. Lane 4: ChABC Sample 2.
Following PCR, ChABC DNA from the Agarose gel was excised and purified and stored at -20C.
Protein Expression
ChABC was ligated into pSB1A3 using XbaI and SpeI, and this expression vector was transformed into competent E.coli BL21 (DE3) cells for protein expression through IPTG-induction at 37C. A SDS-PAGE gel analysis of these expression results are shown below in Figure 3 (lane 2 - 5). Lanes 6 - 9 are samples from expression of a tyrosinase enzyme (BBa_K3794003) and are unrelated to this parts page. In Lane 5 there is a medium-sized band that could potentially be ChABC in the soluble fraction of the E.coli cell samples, however it is not clear nor significant.
There were no distinct, conclusive bands pointing towards expression of 6xHis tagged ChABC from the samples taking during protein expression. A band should appear around the 100-130kDa region to demonstrate presence of ChABC. Despite this, we proceeded to conduct a protein purification using a Qiagen Ni-NTA Spin column under native conditions.
Protein Purification
A Ni-NTA spin column (Qiagen) was used to purify our expressed ChABC protein (which corresponds to BBa_K3794004). Protein purification was conducted under native conditions. However, no conclusive bands that could be inferred to be ChABC (size ~115kDa) could be seen.
Conducting literature reviews following this, we learnt that the isoelectric point of thermostabilised ChABC (pH 6.99) is very similar to the pH of our SDS-PAGE stacking gel (6.8). Heating our ChABC samples prior to loading onto SDS-PAGE gel potentially exposes hydrophobic regions which can cause protein aggregation on the gel. Running a gel with non-heated samples improved our results (Figure 4). Here we can see purified ChABC, albeit in a low yield.
Western Blotting
Following protein purification, a western blot analysis (Figure 5) using an anti-his antibody was conducted to confirm the presence of 6xHis ChABC (BBa_K3794004 - this part is a result of the expression from composite part BBa_K3794005). Western Blot results indicated presence of 6xHis tagged ChABC as shown below. Lanes 2 - 6 all display bands around the ~100kDa band which confirm the presence of 6xHis tagged ChABC.
Protein Expression 2.0
Following a low yield of purified protein, and inconclusive expression results. We used competent BL21 E.coli instead of their DE3 counterparts in an effort to improve soluble protein yield. Following expression using BL21 and induction with IPTG, we ran an SDS-PAGE gel to see the results of our new expression system. As it can be seen below, using BL21 E.coli improved the yield of ChABC, however our protein largely remained in the insoluble fraction in the form of aggregates (as can be seen in Lane 2, 4, 6 and 8). ChABC in the soluble fraction along the protein expression timeline (1 - 3 hours) did not significantly improve. Despite this we moved forward, attempting to purify little soluble ChABC we did have.
Protein Purification 2.0
Having slightly improved the yield of our soluble protein, we conducted another protein purification, however instead using a Ni-NTA resin (ThermoFisher) rather than a Spin column (Qiagen). The results of our second protein purification can be seen below in Figure 7
Similar to previous results, the yield of our purified protein still remained low, especially in the eluted protein samples (lane 7 - 10) (around the ~100kDa range). There are several reasons for this; due to the large size of ChABC (~115kDa), it may be that the Ni-NTA column is getting oversaturated with protein, and unable to accommodate a sufficient yield. Another possibility may be that the mutations we have introduced affect the solubility of ChABC, or that E.coli may not be sufficient hosts for the protein as ChABC is already a bacterial enzyme derived from Proteus vulgaris .
Enzymatic Assay
Despite having a low yield of purified ChABC, we attempted to conduct an enzymatic assay to prove that our recombinant protein is functional at 37C. Following concentration of our eluted protein samples, and a buffer exchange using 15mM Tris-HCl we were able generate a concentrated protein sample of 0.014mg/mLSequence and Features
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