INPN

WHY INP

Ice-nucleation protein (INP), an outer membrane protein from Pseudomonas syringae, is responsible for promoting nucleation of ice at relatively high temperatures (above -50C). The proteins are localised at the outer membrane surface and can cause frost damage to many plants. It is composed of three domains structurally distinguished as the N-terminal domain (191 amino acids, 15% of the protein), which is the portion most responsible for targeting to the cell surface, the C-terminal domain (49 amino acids, 4% of the protein), and the central domain, composed of repeats comprising an 8-, 16-, and 48-residue periodicity that acts as a template for ice crystal formation.

WHY INPNC

INP has been successfully used to display several proteins, such as levansucrase, carboxymethylcellulase (CMCase), Hepatitis B surface antigen (HbsAg), human immunodeficiency virus type 1 (HIV-1) gp120 and so on[1]. This was achieved using either full-length sequences ortruncated portions containing only N- and C-domains (INP- NC) or INP-NC with five additional internal repeating units to display foreign protein on the surface of cell. containing only N- and C-domains means no repeat sequent, producing no ice-nucleation activity. This indicates that the central repeating domains are not required for export to the cell surface, and are therefore, ideal spacer units to control the distance between the passenger protein and the cell surface. Importantly, INP can be expressed at the cell surface of E. coli at a very high level, without affecting cell viability: comparable to the endogenous expression of the OmpA porin[2].

Before us, there was some teams that used INPNC as display system successfully, which is encoded by inaZ. INPNC is used to display passenger protein, such as EYFP, silica binding protein and so on. After researching NCBI, we found that there are several types of nucleotides encoding INP, including inaZ, inaQ, inaK, inaV, inaX. Because inaZ, inaV ,inaX have less support by the published literature comparing to inaQ and inaK, we hope that we can use inaQ or inaK as target gene of recombinant plasmid to alleviate burden of E.coli.

The Story of E.coli Prometheus

BNU-China