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 HER-2, 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 PUREfree 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..

Characterisation

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Conclusion and Perspectives

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References

1. article 1 xxxxxxxx
2. article 2 xxxxxxx