Team:SHSBNU China/Partnership
Partnership
We met iBowu-China in the CCiC Beijing region meeting and had kept in touch with them ever since. They first contact us in September when they have to find a way of purifying their targeted protein because they could not purify their protein by the ordinary Ni-NTA beads-His-tag interaction. Knowing that our team has built a fusion protein that has the potential of using its cellulose binding domains to bind cellulose, thereby purifying the linked domain, in their case, beta-glucuronidase.
Discussion
The student leader of iBowu_China is presenting his project.
The student leader of SHSBNU_China is presenting his project.
After the presentation, we discussed possible aspects that our teams can collaborate with. We first put forward the problem of not having a low enough induction for the inclusion body to reduce the formation and increase the production efficiency of the target protein. Regarding this problem, iBowu_China proposed to let us use their mutated promoters which they have tested to be less strong, in hope of reducing the transcription intensity of our gene. However, due to lack of time for extensive experiments of testing those promoters, we decided to test their promoters for our genes in the future. What we discussed in greater depth is the possibility of using our CBD domains as a new platform for protein purification.
Using CBD domains have two benefits for the purification of iBowu_China’s enzyme:
1. Since the ultimate goal of their project is to use their enzyme in pharmaceutical production, it is necessary to produce great quantities of the enzyme at low costs. With Cellulose Binding Domain, they can purify the enzyme with only Cellulose which costs just $47.5/500g[1], more than 10 times cheaper than the Ni-NTA beads which costs almost $5000/500ml[2].
2. Also, with the flexible linkers, the bG enzyme coded by the sequence, which is actually one monomer of the complete polymer protein, can bind to other monomers that can increase its efficiency greatly.
To solve the problem of the inability of purifying the target protein, we proposed to iBowu_China of using cellulose suspension as a substitute for the ordinary Ni-NTA beads and substitute the His-tag with our CBD domains on each end of the enzyme. We deleted the original Streptavidin domain from the protein and substituted it with iBowu_China’s iBowu_China sent us their gene for us to be added into our cellulose binding platform and to be transformed into Escherichia coli. After we had done the modifications to our cellulose binding platform and added iBowu_China’s sequence in it, we sent it back to iBowu_China’s laboratory for further analysis.
Designed gene circuit with bG enzyme incorporated into cellulose binding platform (BBa_K3716028).
Designed gene circuit with bG enzyme incorporated into cellulose binding platform (BBa_K3716028)
The first trial of the induction of the protein was not successful and no protein was produced. The second trial was carried out with the induction intensity same as what we used in the expression of BBa_K3798032.
Results
Due to lack of time, the purification had not been experimented. However, the efficacy of the fusion protein was examined.
Figure 1 the result of the expression of the fusion protein. a. the visual distinction between the Phenolphthalein-β-D-glucuronide when added expressed cells and the unexpressed cells. b. the absorbance of wavelength 540/600nm
From the result, we can conclude that the beta-glucuronidase fused into our CBD platform is still viable and can perform its job of catalyzing glucuronide. Therefore, this has proven our CBD platform would not distort the function of the original protein that is linked. In the future, we hope to continue the partnership with iBowu_China to finish the purification experiment and make the mass production of glycyrrhetinic acid feasible.
References:
1.Methyl Cellulose, Powder, Laboratory Grade, 500 g. (2021). Retrieved 19 October 2021, from https://www.carolina.com/specialty-chemicals-m-o/methyl-cellulose-powder-laboratory-grade-500-g/875163.pr
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