Part:BBa_K3759019
mLCC-linker-BsLA
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079
Illegal NheI site found at 193 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 261
Illegal EcoRI site found at 1079 - 1000COMPATIBLE WITH RFC[1000]
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-BsLA fusion protein, the PET degradation efficiency will be enhanced enormously, due to the unique properties of amphiphilicity and self-assembly of hydrophobin BslA. Also, as BslA was extracted from bacteria and was a bacterial hydrophobin, it shows a better fusion with mLCC, which help the increment of the PET degradation efficiency of mLCC-linker-BslA.
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.
BslA is a structurally defined bacterial hydrophobin that was found in the biofilm of Bacillus subtilis. It helps the assembling of TasA (an exopolysaccharide and an amyloid fiber-forming protein), the component of the biofilm matrix. BslA is composed of an Ig-type fold with the addition of an unusual, extremely hydrophobic “cap” region. The central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. [3]
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 Purification
Create a fusion protein by jointing mLCC with hydrophobins
Because the pET28a vector has His-tag, therefore we used affinity chromatography to purify the supernatant obtained after bacteria clastogenesis using nickel. The result of our proteogels after elution with 200 mm imidazole and 300 mm imidazole is shown below. We successfully purified mLCC and mLCC-linker-BslA. At the same time, we can also see that the two proteins, mLCC-mHGFI, mLCC-mHFBI, are expressed, but the protein expression level is very low.
Figure 1.(a). M: marker;
Lane 1, 2: mLCC-linker-BslA 43kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 3, 4: mLCC 28kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 5, 6: mLCC-linker-mHFBI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
Lane 7, 8: mLCC-linker-mHGFI 36kDa, elution concentration: 200mm Imidazole, 300mm Imidazole;
PET Degradation Reaction
Create a fusion protein by jointing mLCC with hydrophobins
We perform PET degradation reaction to detect enzyme activity. We set several protein concentration to detect enzyme activity, under the condition of pH8, 70℃, and 18h of reaction time. Then we measured the absorption value at uv240nm by nanodrop, which is the absorption position of the product TPA, and we were surprised to find that the relative enzyme activity of mLCC-linker-BslA was increased about 3 times compared to mLCC!
(a)
(b)
Figure 2. (a). The adsorption value of mLCC, mLCC-Linker-BslA of UV240 under the condition of pH8, 70 celsius degree, reaction time of 18h.
(b) The relativity activity of mLCC, mLCC-Linker-BslA.
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.
[3]: “BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm.” Proceedings of the National Academy of Sciences of the United States of America vol. 110,33 (2013): 13600-5. doi:10.1073/pnas.1306390110
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