Part:BBa_K4768005

split T7 RNA polymerase (Nterm) conjugated to Pertuzumab with a soluble linker

Part for Expression of the split T7 RNA polymerase (Nterm) conjugated to Pertuzumab with a soluble linker in PURE System

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 40
  
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 636
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 40
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 40
  Illegal AgeI site found at 1104
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 21
  Illegal SapI.rc site found at 1740
  

Introduction

Figure 1: Pertuzumab-SL-Nterm structure.

The CALIPSO part BBa_K4768005 is composed of the N-terminal subunit of the T7 RNA polymerase (residues 1 to 180) fused to the anti-HER2 antibody Pertuzumab through a soluble linker. This gene is under transcriptional control of an SP6 promoter and T7 terminator.

This part, coupled to the part BBa_K4768006 containing the C-terminal subunit of the T7 RNA polymerase, has been designed to develop a split T7 RNAP-based biosensor capable of recognizing HER2, an epidermal growth factor that is overexpressed in cancer cells [1], in solution.

The HER2-induced T7 RNAP complex was designed from two existing constructs: a split T7 RNAP-based biosensor for the detection of rapamycin [2] and a split luciferase conjugated with antibodies capable of recognizing HER2 [3]. We decided to merge the relevant functionalities of these two constructs and created a new biosensor that transduces HER2 binding to gene expression activation.

Figure 2: Recognition of HER2 extracellular domain induces functional assembly of the split T7 RNA polymerase, which enables gene expression of target gene under control of a T7 promoter.

Construction

The CALIPSO part BBa_K4768005 consists in the N-terminal subunit of the T7 RNA polymerase fused to Pertuzumab, an anti-HER2 antibody, on its C-terminal domain through an 8-amino-acid linker of glycine and serine residues. The synthesis of this gBlock was made by IDT.

The gBlock was then cloned into the pET_21a(+) plasmid and transformed into Stellar competent cells. Figure 3 shows the restriction profile of the resulting clones. Clone 8 was digested using BsaI. Two bands were expected at 1.3 kb and 5.8 kb. Clones 6 and 15 were digested using EcoRV and XhoI. Two bands were expected at 2.6 kb and 4.6 kb. Only clone 8 showed the expected pattern (lane 3). Plasmids from clones 6 and 15 seemed to be the initial pET_21a(+).

Figure 3: Digestion analysis by BsaI of plasmid extracted from clone 8, and double digestion by EcoRV and XhoI of plasmids extracted from clone 6 and 15.

Production

We first expressed part BBa_K4768005 from its DNA template using the GeneFrontier PUREfrex 2.0 kit supplemented with SP6 RNAP. The reaction products were analyzed by SDS-PAGE. Because the theoretical molecular weight is 69 kDa, no other band from PURE system proteins was expected to migrate at this size. The protein pattern shown in Figure 4 exhibits an additional band around 69 kDa compared to the negative controls. This result indicates successful production of the full-length Pertuzumab-SL-Nterm in PUREfrex 2.0.

Figure 4: SDS-PAGE (10% polyacrylamide) analysis of Pertuzumab-SL-T7Nterm visualized by Instant Blue staining. The arrowhead indicates the additional band corresponding to Pertuzumab-SL-T7Nterm (69 kDa). PUREfrex2.0 was used. SP6 RNAP was added in the negative control (T-). In the positive control (T+), DHFR was expressed but the band ran out of the gel due to the low molecular weight.

Next, we produced Pertuzumab-SL-Nterm using the PUREfrex 2.1 kit to promote disulfide bond formation due to non reducing conditions. SDS-PAGE analysis shows the expected band of the protein at 69 kDa (Figure 5).

Figure 5: SDS-PAGE (10% polyacrylamide) analysis of Pertuzumab-SL-T7Nterm visualized by co-translational labeling with GreenLys. The arrowhead indicates the additional band corresponding to Pertuzumab-SL-T7Nterm (69 kDa). PUREfrex2.1 was used. SP6 RNAP was added in the negative control (T-). In the positive control (T+), DHFR was expressed but the band ran out of the gel due to the low molecular weight.

Conclusion and Perspectives

We can conclude that the recombinant protein Pertuzumab-SL-T7Nterm can be cell-free expressed in both reducing PUREfrex 2.0 and non reducing (disulfide bond promoting) PUREfrex 2.1. It is worth mentioning that the sequencing results obtained after we performed these experiments showed a 675-nucleotide deletion within the region of the heavy chain of Pertuzumab. It remains to be investigated whether this deletion originated during the PCR that preceded the cloning or at the sequencing stage.

Due to unsuccessful cloning of the second part of the biosensor, Trastuzumab-SL-T7Cterm, the functionality assays could not be performed. We encourage future iGEM teams to attempt other cloning strategies, pursue the characterization of the biosensor, and to contact us for further details. This construction can be manipulated in a biosafety level 1 laboratory.

References

1. Jois et al. 2021. Peptidomimetic Ligand-Functionalized HER2 Targeted Liposome as Nano-Carrier Designed for Doxorubicin Delivery in Cancer Therapy. Pharmaceuticals. 14(3). 221
2. Pu, J., Zinkus-Boltz, J., Dickinson, B. C. 2017. Evolution of a split RNA polymerase as a versatile biosensor platform. Nat Chem Biology 13(4). 432-438.
3. Stains, C. I., Furman, J. L., Porter, J. R., Rajagopal, S., Li, Y., Wyatt, R. T., Ghosh, I. 2010. A General Approach for Receptor and Antibody-Targeted Detection of Native Proteins utilizing Split-Luciferase Reassembly. ACS Chem Biol 5(10). 943-952