See BBa_K811003 for details. INPNC with 5' BamHI and 3' PstI cloning sites on the C terminus with intervening GS linker domain. This construct can be used for the surface display of large proteins by ligating your gene of interest between the BamHI and PstI cut sites. To design a forward PCR primer compatible with this biobrick, please use the following attachment as a 5' overhang: AGGCGGATCCGGT-GENE. This will include the BamHI cut site and will also ensure that your insert is in-frame with the INPNC-GSlinker so that it will properly be displayed on the surface.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BamHI site found at 952
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 72
Illegal NgoMIV site found at 405
Illegal AgeI site found at 823
- 1000COMPATIBLE WITH RFC
Usage and Biology
Ice nucleation protein (INP) is a protein found in Xanthomonas campestris pc. campestris BCRC 12846. It functions as, as its namesake suggests, causing ice nucleation and formation. However, recent studies have utilized INP for its surface display properties. In nature, the protein is anchored in the membrane through a glycosylphosphatidylinositol (GPI) anchor, a relatively rare occurance in prokaryotes.
The INP protein is composed of a N-terminal region that appears to interact with the phospholipid membrane, a C-terminus hydrophillic region that is exposed to the outside membrane, as well as a central 8, 16, or 48 amino acid motif that is responsible for INP's ice nucleation properties. However, this central amino acid motif is not necessary for INP's surface display properties. Therefore, scientists truncated the proteamin, retaining only the N (179 aa) and C termini (49 aa) to produce INPNC.
This truncated protein retains INP's membrane display abilities, and also contains a GS amino acid linker followed by a site containing multiple restriction sites for the easy ligation of additional DNA for INPNC fusion experiments and surface display of desired proteins.
Confirmation of functionality of BBa_K811005
We used a modified form of BBa_K811005 in Kyoto 2016’s project and added further characterization to this part.
First, we constructed INPNC-His-scFv encoding plasmid. scFv we used as a passenger protein to this surface expression domain was anti-Norovirus scFv. It specifically binds to Norovirus(NoV). We transformed it into E. coli strain DH5α to examine the physical interaction between the transformants and NoV-like particles (NoVLPs, the capsid proteins of norovirus). By scanning electron microscopy, we clearly observed NoVLPs bound on the surface of INPNC-His-scFv expressing E. coli cells. This, along with other results in our project show that a protein of interest can be expressed on the surface of E. coli by this INPNC module to capture target molecule(s). This visual demonstration on the function of this parts is shown in our project.
Addition of His-tag to BBa_K811005
This addition introduces two new functions to the fusion protein:
1) We can use anti-His tag antibody to detect their surface expression regardless of passenger protein placed downstream.
We confirmed this with detections of the INPNC-His-(passenger protein) by Western blotting using anti-His tag antibody.
2) INPNC-His-(passenger protein) can be purified using nickel columns.
Taking advantage of this His-tag system, we can isolate our fusion proteins from the insoluble membrane fractions of E. coli cells, after solubilizing with 7M guanidine-HCl. This would allow fusion protein detection even if the protein’s expression levels are low, further strengthening the first function.
We tested and confirmed this function. See our project for more details.