Improvements
Group: BJEA_China 2021
Author: Kairuo Zhang
Summary: Hydrophobins are small surface-active proteins and have both fungal and bacterial origins. Hydrophobins originated from fungi are divided in to two class and are being widely used and applied. HGFI[1] and HGFI[2] are hydrophobins derived from fungi, HGFI is a class I hydrophobin, and HFBI is a class II hydrophobin. Hydrophobin’ s hydrophobic part is exposed on the surface forming a planar area called the hydrophobic patch, making the surface of the protein contain both hydrophobic and hydrophilic area therefore making the surface making the hydrophobins amphiphilic. Because the hydrophobins’ amphiphilicity property it can self-assemble themselves with others. In 2015, the team Tianjin mutated this hydrophobin, and the resulting mHGFI (inJanus-m, BBa_K1582002 ) and mHFBI ( sJanus-m, BBa_K1582003 ) can be expressed in bacteria.
Recently, a research published on Nature came up with a mutant enzyme, mLCC[3] 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.
We construct the fusion protein which was made to enhance the efficiency of adsorption since the surface of PET film is hydrophobic and the surface of mLCC is hydrophilic. By constructing the mLCC-linker-mHFBI( BBa_K3759022 ) and mLCC-linker-mHGFI( BBa_K3759018 ) and fusion protein, the PET degradation efficiency will be enhanced due to the unique properties of amphiphilicity and self-assembly of hydrophobins.
Protein Expression
(Figure 1. The expression of mLCC(Left 3rd 4th)，mLCC-linker-mHGFI(Left 5th 6th), mLCC-linker-mHFBI(Left 7nd 8th))
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 3rd, 5th, 7th aimed protein, and we used 300mM imidazole to eluting the left 4th, 6th ,8th aimed protein.
References
[1] J Vereman, Thysens T , Derdelinckx G , et al. Extraction and spray drying of Class Ⅱ hydrophobin HFBI produced by Trichoderma reesei[J]. Process Biochemistry, 2019, 77(FEB.):159-163. [2] Song D , Wang X , Gao Z , et al. Expression, Purification and Characterization of Hydrophobin HGFI from Grifola Frondosa in Saccharomyces Cerevisiae[J]. Acta Scientiarum Naturalium Universitatis Nankaiensis, 2018. [3] 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.
