Part:BBa_K5416001
HRT2-SP
This part is designed by Team Imperial_College in iGEM 2024. As part of their VLP design (BBa_K5416060)
This basic part is a variant of HRT2trunc (BBa_K5416000), and codes for a truncated version of HRT2 rubber synthase enzyme (BBa_K1088016). Comparing to HRT2trunc, the His-tag has been replaced with a P22 scaffold protein (BBa_K3187021) to mediate binding towards the inner surface of P22 bacterial phage virus-like-particle (VLP).
Please be advised this part works the best as a composite part: BBa_K5416062
We have shown this part to have the following functions:
- Produces Rubber in small quantity
- Interacts with P22 VLP capsid to be encapsulated
Design
We designed HRT2-SP based on the structural information of HRT2trunc, which is also engineered in this project. This enzyme, in short, contains the major catalytic unit of the rubber synthase HRT2 for rubber production, whereas the dimerizing helice has been replaced with a scaffold protein to allow integration into a VLP [1][2]. This process of protein engineering is carried out in silico through using advanced modeling methods, in which is documented with great detail in here.
Fig 1: An illustration of how this part is designed
Fig 2: HRT2-SP binds to the inner surface of P22 through structural alignment with a known P22-SP complexed structure.
Characterization
We have characterized this part in its composite form BBa_K5416060
Expression
Fig 3: SDSPAGE analysis result of IPTG-induced E. coli cell (BL21(DE3)) transformed with this part and expressing P22 at the same time. Cells are induced and cultured 16hr at 25C. From left to right: lane L: Transgen 10-180kDa protein marker; lane 1: cell pellet after lysed with BugBuster with 0.2mg/ml lysozyme for 30min at room temperature; lane 2: lysate supernatant centrifuged at 3k rpm for 10min; lane 3: lysate supernatant centrifuged at 12k rpm for 10min; lane 4: cells sampled directly from the culture media; lane 5: 0.45um PDMV-filtered lysate (after centrifuged at 3k rpm); lane 6: first wash (with PBS) after protein loaded to Ni-NTA columns; lane 7: second wash (PBS with 2mM imidazole) of the Ni-NTA column; lane 8: 5ul of 20x concentrated elute (200mM imidazole); lane 9: 80x concentrated lysate supernatant, 5ul. All lanes except lanes 8 and 9 were loaded with 10ul of samples. The P22 capsid protein around 46kDa and HRT2trunc protein around 25kDa were identified with red arrows in the gel.
Rubber Production
Fig 4: Upper image: The UV-vis absorbance spectrum of the natrual rubber extracted from cyclohexane for E. coli expressing this part (6-blank), comparing to a negative control (pET-blank, pET28a transformant). Lower image: comparision of A210 reading of E. coli expressing this part with negative control (pET28a strain), and a known postive control which 10ug of polyisoprene is added to the negative control. All absorbance values are blanked with absorbance spectrum of cyclohexane.
References
1. Takahashi S, Koyama T. Structure and function of cis‐prenyl chain elongating enzymes. The Chemical Record. 2006;6(4):194-205.
2. McCoy K, Selivanovitch E, Luque D, Lee B, Edwards E, Castón JR, Douglas T. Cargo retention inside P22 virus-like particles. Biomacromolecules. 2018 Aug 9;19(9):3738-46.
3. Mirdita M, Schütze K, Moriwaki Y, Heo L, Ovchinnikov S, Steinegger M. ColabFold: making protein folding accessible to all. Nature methods. 2022 Jun;19(6):679-82.
4. Xiao H, Zhou J, Yang F, Liu Z, Song J, Chen W, Liu H, Cheng L. Assembly and capsid expansion mechanism of bacteriophage P22 revealed by high-resolution cryo-EM structures. Viruses. 2023 Jan 26;15(2):355.
5. Bittrich S, Segura J, Duarte JM, Burley SK, Rose Y. RCSB protein data bank: Exploring protein 3D similarities via comprehensive structural alignments. Bioinformatics. 2024 Jun 13:btae370.
6. Salvucci ME, Coffelt TA, Cornish K. Improved methods for extraction and quantification of resin and rubber from guayule. Industrial Crops and Products. 2009 Jul 1;30(1):9-16.
7. Asawatreratanakul K, Zhang YW, Wititsuwannakul D, Wititsuwannakul R, Takahashi S, Rattanapittayaporn A, Koyama T. Molecular cloning, expression and characterization of cDNA encoding cis‐prenyltransferases from Hevea brasiliensis: a key factor participating in natural rubber biosynthesis. European Journal of Biochemistry. 2003 Dec;270(23):4671-80.
Index
MIPTHIAFIL DGNGRFAKKH KLPEGGGHKA GFLALLNVLT YCYELGVKYA TIYAFSIDNF RRKPHEVQYV MNLMLEKIEG MIMEESIINA YDICVRFVGN LKLLDEPLKT AADKIMRATA KNSKFVLLLA VCYTSTDSPI TGDVSAANKD AIRKQMDAAA SKGDVETYRK LKAKLKGIRS SSGINPYPDV LIRTSGETRL SNYLLWQTTN CILYSPHALW PEIGLRHVVW AVQ
The sequnence above is the amino acid sequenc of this part. Displayed here to make codon optimization and protein engineering easier. The sequence in deep green (html#003300) codes for the truncated major enzyme unit of the HRT2 and HRT2trunc. The sequence in yellow (#ffff00) is the scaffold protein domain.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
None |