Part:BBa_K5357016
PelB-anti-Cystatin C Nanobody-(GGGGS)3 linker-NanoLuciferase-GGSG linker-6xHis tag Coding Sequence
Logic of Assembly
This composite part encodes a fusion protein of PelB-anti-Cystatin C nanobody-(GGGGS)3 linker-NanoLuciferase-GGSG linker-His tag. PelB (see BBa_K5357003) is a secretion signal that ensures localisation of the protein in the periplasmic space of E. coli, a reducing environment that enables proper disulphide bond formation. PelB is cleaved off in the periplasm.
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. The final product is a correctly folded anti-Cystatin C Nanobody-NanoLuciferase protein conjugate, which can be purified by leveraging its 6xHis tag in IMAC (immobilised metal affinity chromatography).
An alphafold 2 model of nanobody-linker-NanoLuciferase (NB-NL).
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 moloclonal 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 NanoLuciferase 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. There were two questions we asked about this conjugate: 1. Does it still retain the bioluminescent activity of NanoLuciferase, and 2. Does it still retain the Cys C binding capabilities of the anti-Cys C nanobody?
Design Notes
The coding sequences were taken from multiple sources (see 'Source' section). The anti-Cys C nanobody sequence we used was NB44 from Mi 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 part as a gBlock from GenScript.
Characterisation
Using Golden Gate assembly, this gBlock was ligated into the pSB1C3 plasmid. The plasmid was then transformed into E.coli DH5 alpha cells and cultured on agar media, which contained chloramphenicol to select for transformed cells. We used blue/white screening to visualise colonies containing pSB1C3 plasmids with the gBlock inserted. All colonies appeared white, indicating they had the plasmid with the insert.
We performed a diagnostic digest on our plasmids. The fragments were the correct size, corresponding to the molecular weight of the pSB1C3 plasmid backbone and our gBlock (10th and 11th lanes from the left).
We transformed BL21 cells and carried out protein expression, but no protein was expressed (no bands in the NB-NL lanes).
We therefore sent a sample of our plasmids to our sponsor FullCircle Labs, who sequenced them. We found from these results that our plasmids were concatamers, containing multiple insertions of the NB-NL gBlock. The reason the diagnostic digest did not indicate this is because we used restriction enzymes that cut within the gBlock sequence.
We selected different colonies of DH5 alpha and did miniprep to extract the plasmids. We sent these for sequencing, and found that they were the correct size and were not concatamers.
The plasmids were then transformed into E. coli BL21 cells to express the protein. The cells were cultured on chloramphenicol to select for those that were transformed.
We extracted the protein from the cells via periplamic extraction with TES buffer, and purified the protein via IMAC. We used buffer exchange to get rid of imidzole from the protein solution. We did a Bradford assay to quantify the concentration of the protein, which was 5.5 ug/ml. We carried out a western blot and confrimed the size of our NB-NL was correct (lane 2 contains NB-NL, whose size is consistent with that of the size of the protein (band at 35 kDa)).
Enzymatic Assay
To assess whether nanobody-NanoLuciferase (NB-NL) maintains the bioluminescent activity of NL, we carried out an enzymatic assay by measuring the bioluminescence after having provided the NB-NL or control NL with the NanoGlo luciferase assay, which contains the substrate furimazine. Our assay data pointed at the possibility that NB-NL is more active than NL at a lower concentration, something which we found surprising:
Additionally, when we introduced NanoGlo into the wells containing NB-NL, bioluminesence was highly visible:
ELISA assay
We performed an ELISA assay to determine whether the NB domain in NB-NL still retains its Cystatin-C binding capabilities, and found there to be a significant difference between the control (where NL and NanoGlo (NG) were introduced into the wells with immobilised Cys C) and the test sample (where NB-NL and NG were introduced into the wells with immobilised Cys C), indicating the likelihood of NB-NL staying in the wells post-wash step is significantly different to that of NL. This means the immobilised Cys C in the wells captures and binds to NB-NL strongly enough that there is significantly higher bioluminescence between the wells containing NB-NL and those containing NL. This indicates that the NB domain in NB-NL does retain its Cystatin C binding capabilities.
Shown below are (1) the control bioluminescence graph (upper) and (2) the test bioluminescence graph (lower).
All bioluminescence values are averaged over three identically prepared samples. The graphs display 5% error bars and bioluminescence readings are taken once every 30 seconds.
In conclusion, NB-NL retains the activity of NL, and may actually exceed it. Our preliminary studies provide significant evidence that NB-NL can bind Cys C via its NB domain. We hope these findings have paved the way for new diagnostic technologies harnessing NB-NL conjugates for the detection of biomarkers. For more details on these findings, please see our proof of concept page (wiki).
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
Sequence and Features
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