Part:BBa_K811004
INPNC-HA
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 72
Illegal NgoMIV site found at 405
Illegal AgeI site found at 823 - 1000COMPATIBLE WITH RFC[1000]
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 protein, 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 C-terminus HA tag suitable for detection via antibodies.
Characterization
INPNC (BBa_K81103) was tagged with an HA tag on the C terminus to verify surface expression. This construct was cloned into the pET-26b(+) vector and then expressed in BL21 cells after induction with 1mM IPTG. The cells were incubated with anti-HA antibody conjugated to Alex Fluor 647 dye from Cell Signalling Technologies and imaged with a confocal microscope (Figure 1).
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