HRT2-SP

Imperial-College 2024

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

The diagram above annotated the domains of this part in different colors: Major catalytic unit (green) and scaffold protein (yellow), wHihc has been identified from the Alphafold2-predicted structure after analysing in Pymol ^[3]. We then asked if this folded structure would be able to interact with the capsid protein of P22 VLP, for which the SP domain is aligned and compared with a known structure where the SP binds to the P22 capsid ^[4][5].

Fig 2: HRT2-SP binds to the inner surface of P22 through structural alignment with a known P22-SP complexed structure.

The alignment result shows very high similarity of the SP domain of the HRT2-SP (pink) with the known structure(light blue). And thus in dry lab it is evidenced that the protein protein interaction of this fusion protein exists with the P22 capsid.

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.

The gel above shows successful expression of this part inside E. coli. Indicated by the band around 25kDa. Following the lysis of the cells via BugBuster solution with 0.2mg/ml lysozyme (Sigma-Aldrich, UK), this part persistently to exist in the liquid phase regardless of high-speed centrifugation (both in after spinning at 3k and 12k rpm). This indicates the high solubility of the VLP. The lysate was then passed through a 0.45um filter to remove insoluble proteins and cell debris and loaded into Ni-NTA column for His-tag specific protein purification. It was observed that minimal amount of targeted protein was existing in the flowthrough of washing steps (with PBS and low concentration of imidazole), which suggesting the binding of the VLP proteins to the nickel ions via the his-tag. This hypothesis was further evidenced by identifying good amount of VLP proteins after the column is washed with imidazole (lane 8). This elute is concentrated 20 times by volume using a 30kDa filter spin column(Merck, USA). Noticeably, even the HRT2-SP protein does not carry any His-tags and is smaller than threshold (<30kDa) of the spin column filter. The band for HRT2-SP has also been identified in the same lane, which provides strong evidence that the HRT2-SP could only be trapped inside the VLP to resist the washes.

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.

The rubber producing ability of this part is assessed with 100ml of culture of the transformant, where the natural rubber produced by this part is extracted with cyclohexane following the reported protocol ^[6][7]. The characteristic absorbance at 210nm is compared with the wild-type BL21 (pET28a transformant), and known concentrations of cis-polyisoprene, that was determined to be approximately 10ug per 100ml.

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.

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Sequence and Features

Assembly Compatibility: