Part:BBa_K3759022
mLCC-linker-mHFBI
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 199
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 1016
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1025
Usage
It has been well known that the surface of PET film is hydrophobic, and the surface of mLCC is hydrophilic. By constructing the mLCC-linker-mHFBI fusion protein, the PET degradation efficiency will be enhanced enormously, due to the unique properties of amphiphilicity and self-assembly of hydrophobin mHFBI.
Biology
LCC is a leaf-branch compost cutinase[1] and a kinetically robust protein[2]. A research published on Nature came up with a mutant enzyme, mLCC[1] that hydrolyzes 90% of PET in plastic bottles in just 10 hours. This is more efficient than any previous PET hydrolase, and more importantly, the resulting monomers- ethylene glycol and terephthalic acid have the same properties as the monomers found in petrochemical materials.
The linker is GGGGSGGGGS.
Hydrophobin could be produced by filamentous fungi, such as Ascomycetes and Basidiomycetes. Many different aspects of fungal development have been attributed to hydrophobin. For example, they are thought to play a role in the formation of aerial hyphae and fruiting bodies. One of the most important features of hydrophobin is that they are able to assemble spontaneously into amphipathic monolayers at hydrophobic–hydrophilic interfaces. There are two classes of hydrophobin, which are divided by the stability of their self-assembly. HFBI from Trichoderma reesei belongs to Class II. The assemblies of class II hrdrophobin can be dissolved in ethanol or sodium dodecyl sulfate or through the application of pressure or lowering of the temperature. Team_Tianjin 2015 did some mutations to it, so it can be expressed in E.coli.
Design Consideration
The construct was cloned into a pET28a plasmid and transformed into BL21 (DE3) E. coli.
The construction includes:
1. a 6× His tag is added to enable us carrying out Ni-NTA protein purification.
Protein Expression
Figure 1. The expression of mLCC(Left 3rd 4th) and mLCC-linker-mHFBI(Left 7th8th)
Pre-expression:
The BL21 bacteria that contains aimed protein were cultured in 5mL LB liquid medium with kanamycin in 37℃ overnight. After taking samples, we transfer them into 1L LB medium with kanamycin.
Cultured in bottles:
After culturing in 37℃ in bottles, we used 0.5mM IPTG induced in 16℃ for 24 hours. Then, we used 200mM imidazole to eluting and get left 7th aimed protein, and we used 300mM imidazole to eluting the left 8th aimed protein.
References
[1] Tournier, V. , Topham, C. M. , Gilles, A. , David, B. , & Marty, A. . (2020). An engineered pet depolymerase to break down and recycle plastic bottles. Nature, 580(7802), 216-219.
[2] Sulaiman S , You D J , Kanaya E , et al. Crystal Structure and Thermodynamic and Kinetic Stability of Metagenome-Derived LC-Cutinase[J]. Biochemistry, 2014, 53(11):1858-1869.
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