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.

WHY INPN

Then we found that using INP derivatives containing only N-domain (INPN) as display system successfully is possible. The passenger proteins are also various, such as Japanese Encephalitis Virus Pathogenicity, phosphate-binding protein and so on. There is also literature which alleges that since full-length INP is quite large (1,200–1,500 amino acid residues), functional truncated INP molecules may serve as better anchoring motifs to carry large heterologous proteins.

So we start thinking that we also could use INP derivatives containing only N-domain (INPN) as display system. As we say early, inaZ has less support. So, we use inaK and inaQ as ingredient. According to the results of literature survey, inaK-N has about 658bp and inaQ-N has about 525bp. We hoped truncated INP molecules serve as better anchoring motifs to carry heterologous proteins, our ModA.

Reference

[1] Mei Li Wu, Chun Yung Tsai& Tsai Hsia Chen, 2005, Cell surface display of Chi92 on Escherichia coliusing ice nucleation protein for improved catalytic and antifungal activity

[2] Edwin van Bloois, Remko T. Winter, Harald Kolmar and Marco W. Fraaije, 2010, Decorating microbes: surface display of proteins on Escherichia coli.

[3] jinalin Dou, Janet Daly, Zhiming Yuan, Tao Jing, and Tom Solomon,2009,Bacterial cell surface display: a method for studying Japanese Encephalitis virus pathogenicity

[4] Qianqian Li, Ziniu Yu, Xiaohu Shao, Jin He & Lin Li, 2009,Improved phosphate biosorption by bacterial surface display of phosphate-binding protein utilizing ice nucleation protein

The Story of E.coli Prometheus

BNU-China