Part:BBa_K5357022
Contents
Logic of Assembly
This part encodes a PelB-anti-Cystatin C nanobody-(GGGGS)3 linker-NanoLuciferase-(GGGGS)3 linker-NanoLuciferase-GGSG linker-6xHis tag.
PelB (see BBa_K5357003) is a secretion signal that localises the protein to the periplasm of E. coli, which is a reducing environment that facilitates disulphide bond formation, ensuring the nanobody and NanoLuciferases are properly folded into their functional, enzymatically active and binding states. PelB is cleaved off in the periplasm. The final product is what is hopefully a correctly folded nanobody- NanoLuciferase-NanoLuciferase (NB-NL-NL) conjugate, which can be purified using its 6xHis tag (see BBa_K5357002) via immobilised metal affinity chromatography (IMAC).
We used GS linkers because the presence of hydrophobic (G) and hydrophilic (S) amino acids prevents secondary structure formation of the linker, reducing the risk of the linker interfering with the correct folding of the proteins it is connecting.
Usage and Biology
NanoLuciferase is an engineered form of luciferase that luminesces with a significantly higher intensity than luciferase when exposed to its synthetic substrate furimazine. It is smaller (19.1kDa) than normal luciferases (62kDa), which decreases its influence on structure when fused to other proteins. It is also more stable in urea than luciferase. These aspects of NanoLucuiferase made us theorise that it would serve as a good reporter for urinary biomarkers.
Nanobodies are small antibody fragments that share comparable binding abilities as regular antibodies, bind in a wide pH range, and are less likely to aggregate than monoclonal antibodies. They also do not require glycosylation, meaning they can be expressed in cheaper systems like E. coli, instead of mammalian cells.
We therefore designed this part, which encodes two nanoluciferases fused to an anti-Cystatin C nanobody. Cystatin C (Cys C) is a urinary biomarker which is predictive of acute kidney injury (AKI), sepsis, and mortality. In designing this part, we aimed to create a reporter for the detection of Cys C.
By using two NanoLuciferases, we wanted to test if this would increase the bioluminescent intensity of our protein conjugate. If it does, this could pave the way for using tandem NanoLuciferases as reporters instead of single NanoLuciferases.
Design Notes
The coding sequences were taken from multiple sources (see 'Source' section). The anti-Cys C nanobody sequence we used was NB44 from Wang et. al's publication, because this nanobody had the highest association constant to Cys C. The gBlock's sequence was codon-optimised for expression in E. coli, and in places where there were consecutive identical amino acids like in the His tag, synonymous codons were used to prevent ribosomal stalling. We used Benchling to assemble the coding sequences together, and ordered the PSB1C3 plasmid with the composite part already inserted from from Genscript.
Characterisation
We chose not to transform these plasmids into DH5 alpha cells because the plasmids were already provided for in sufficient quantities by Genscript. We instead transformed the plasmids into BL21 cells straight away and these grew. We selected for transformed cells using chloramphenicol. We did not choose to send the plasmids for sequencing because the plasmids were assembled with the coding sequence of NB-NL-NL inserted by Genscript.
We used periplasmic space protein expression IMAC for protein purification. After protein purification, buffer exchanged was performed, replacing the imidazole-rich elution buffer with binding buffer which has minimal concentration of imidazole.
We then performed SDS-PAGE to confirm the size of the protein. A dark band was observed at approximately 60 kDa, which is roughly equal to the molecular weight of NB-NL-NL (54.7 kDa). We then performed a Bradford assay, and determined the concentration to be between 182.58-944.25 ug/ml.[
Enzymatic assay
We wished to compare the bioluminescence of NB-NL to that of NB-NL-NL. We theorised that since NB-NL-NL has twice the number of NL domains compared to NB-NL, it has double the bioluminescence as NB-NL. To test this, we compared the bioluminescence of NB-NL-NL at a specific molar concentration to that of the bioluminescence of NB-NL at half the molar concentration. This ensured that between the two samples, there are an equal number of NL domains, meaning that the differences between the bioluminescence of these samples can only be ascribed to the structural differences between the protein conjugates.
We found that for all concentrations tested, NB-NL-NL, despite having the same number of NL domains as NB-NL, has a comparatively lower bioluminescence. This indicates that the structure of NB-NL-NL prevents it from having as high of a bioluminescence as NB-NL, indicating that using tandem fused NanoLuciferases is not as effective for use as reporters as a single NanoLuciferase.
ELISA Assay
We next wished to determine if we could prove that the NB-NL-NL conjugate binds to Cys C. Whereas the NB-NL conjugate (BBa_K5357016) we designed was demonstrated to bind to Cys C, comparatively, there was less evidence for the binding of NB-NL-NL to Cys C. This is probably either because of the impaired bioluminescence of NB-NL-NL, or the potential misfolsing of NB, or both (see our wiki, proof of concept section for more details).
We hope to have contributed to the efforts of future iGEM teams that work with NanoLuciferase as a reporter, and have provided useful information on the bioluminescent and biomarker binding capabilities of tandem fused NLs compared to single NL. We also hope to inspire future teams into further refining and investigating the binding capabilities of our conjugate.
Source
Pel B: http://www.signalpeptide.de/index.php?sess=&m=listspdb_bacteria&s=details&id=194499&listname=
NL: https://nanolight.com/content/nanoluc-sequence/
anti-Cys C nanobody: Mi L, Wang P, Yan J, Qian J, Lu J, Yu J, et al. A novel photoelectrochemical immunosensor by integration of nanobody and TiO(2) nanotubes for sensitive detection of serum cystatin C. Anal Chim Acta. 2016;902:107-14.
T7 promoter, T7Te terminator, and RBS from the PSB1C3 plasmid sequence (BBa_K2842666).
References
Sockolosky JT, Szoka FC. Periplasmic production via the pET expression system of soluble, bioactive human growth hormone. Protein Expr Purif. 2013;87(2):129-35. Schafer F, Romer U, Emmerlich M, Blumer J, Lubenow H, Steinert K. Automated high-throughput purification of 6xHis-tagged proteins. J Biomol Tech. 2002;13(3):131-42.
Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, et al. Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate. ACS Chem Biol. 2012;7(11):1848-57.
Krasitskaya VV, Efremov MK, Frank LA. Luciferase NLuc Site-Specific Conjugation to Generate Reporters for In Vitro Assays. Bioconjug Chem. 2023;34(7):1282-9.
Mi L, Wang P, Yan J, Qian J, Lu J, Yu J, et al. A novel photoelectrochemical immunosensor by integration of nanobody and TiO(2) nanotubes for sensitive detection of serum cystatin C. Anal Chim Acta. 2016;902:107-14.
Muyldermans S. A guide to: generation and design of nanobodies. FEBS J. 2021;288(7):2084-102. Arbabi-Ghahroudi M. Camelid Single-Domain Antibodies: Historical Perspective and Future Outlook. Front Immunol. 2017;8:1589
| None |