Part:BBa_K4658004

NanoLuc luciferase fusion protein

Our project, (LuciPEP), aimed to develop a method for detecting microplastics in water samples through the strategic design and utilisation of fusion proteins. The first of these fusion proteins was meticulously crafted based on a polypropylene binding anchor peptide (LCI) from previous research (Ji et al., 2021). It incorporates NanoLuc luciferase (NLuc) as a reporter molecule. Here, we go over the characterisation of our NLuc fusion protein, from its successful cloning to its use in a polypropylene binding assay.

Design

The fusion protein incorporates NLuc luciferase (BBa_K1159001), a bioluminescent enzyme taken from the marine shrimp Oplophorus gracilirostris. This is linked to the LCI anchor peptide (BBa_K4658003) with a linker sequence (BBa_K4658000). A TEV cleavage site (BBa_J18918) and an AviTag (BBa_K4658002) with an AviTag spacer (BBa_K4658001) are also included (Fig. 1). The TEV cleavage site allows for the separation of the anchor peptide from the reporter protein following purification, while the AviTag was included for biotinylation of the separated anchor peptide in the assay design. All sequences were codon optimised for expression in E. coli.

Figure 1: A schematic of the fusion protein. NLuc is shown in light blue and it is linked to the LCI anchor peptide shown in yellow. The linker sequence is seen in pink and the AviTag in dark blue.

Modelling

After completing our NLuc fusion protein design, effort was taken to establish a precise computational representation of the fusion protein construct we aimed to engineer. Given the potential for unforeseen epistatic effects in fusion proteins, obtaining an atom-level three-dimensional structure that aligned with our engineered sequences was paramount. In the absence of experimental structures, we utilised the deep-learning model OmegaFold for structural prediction (Fig. 2). Using this predicted structure, we embarked on a two-step molecular dynamics simulation process in order to obtain a more realistic representation of the protein's conformation within its natural environment (Fig. 3).

Figure 2: The structural prediction of our NLuc fusion protein. In copper you can see NLuc luciferase and in yellow the anchor peptide. These are joined by a linker in blue.

Figure 3: The solubilised protein. In copper you can see NLuc luciferase and in yellow the anchor peptide. These are joined by a linker in blue.

Usage and Biology

The detection and quantification of microplastics is crucial for understanding their impact and developing effective mitigation strategies. To overcome this problem of detection and quantification of microplastics in water bodies, we devised an assay where the anchor peptide (BBa_K4658003), which can easily bind a polypropylene surface, along with luciferase for the detection of microplastics with bioluminescence. The assay consists of three parts. First, is the biotinylated anchor peptide which is attached to a streptavidin magnetic bead. Second, is the microplastic which gets captured by the anchor peptide and third, is the NLuc fusion protein which is used for detection. The oxidation of furimazine by NLuc gives off a luminescent signal that can be detected with smartphone technology.

Characterization

Agarose Gel

The cloning of our fusion protein into its vector backbone (pET45b) was successfully carried out using restriction digestion with HindIII and SacI restriction enzymes. The ligation product was transformed into E. coli Top10 cells and cloning was confirmed via colony PCR and sequencing (Fig. 4).

Figure 4: Agarose Gel of the colony PCR confirming successful cloning of the NLuc fusion protein and the eGFP fusion protein (BBa_K4658005).

SDS-PAGE

Protein expression and purification was carried out using E.coli BL21 cells, which was induced with 0.5mM IPTG until the OD reached 0.5-0.6, followed by overnight induction at 25°C and 37°C. The protocols can be found (here). The SDS page for the following is shown below in Fig. 5.

Figure 5: SDS-PAGE showing successful purification of the NLuc fusion protein (BBa_K4658004).

Binding assay

The purified NLuc fusion protein was tested to ascertain binding specificity as well as functionality of the luciferase domain. This was done by analysing the luminescence of bound proteins on a polypropylene 96-well plate with the help of a plate reader. A concentration gradient was tested alongside a blank containing no protein (Fig. 6).

Figure 5: The luminescence intensity was measured at different concentrations (µg/µl) pre-wash (A) and post-wash (B). Pre-wash luminescence values are higher than that of the post wash values. However, post-wash luminescent signal increases following a threshold protein concentration of 1µg/µl. This suggests the successful binding of our fusion protein to polypropylene.

Sequence and features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NotI site found at 232
  Illegal NotI site found at 247
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 291
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 207
  
• 1000
  COMPATIBLE WITH RFC[1000]