Part:BBa_K3984004

N-terminal domain of ice crystal nucleoprotein (INP-N)

The N-terminal domain of ice crystal nucleoprotein (INP-N) can carry passenger proteins and anchor on the surface of bacterial cell membranes.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Application of ice crystal nucleoprotein and cell surface display technology

Microbial cell surface display system refers to the method of combining molecular cloning technology and enzyme engineering technology to combine the gene sequence of the exogenous functional protein with the gene sequence of the anchor site on the surface of the specific receptor microbial cell membrane or cell wall. The fusion protein can be secreted in cells and can recognize specific cell surface structures, so that it can be directly expressed on the surface of host cells in order to achieve practical application value. The cell surface display system is generally composed of carrier protein, passenger protein, and recipient cells.

Carrier proteins are mainly responsible for connecting specific target proteins in the cell surface display system, so that the newly synthesized whole-cell catalyst has special functions. Due to the differences in the structural composition of the recipient cells, the types of carrier proteins are also very different. For example, in the yeast surface display system, the application of the cell wall carrier protein α-lectin protein subunit has become increasingly widespread. Chen et al. used the N-terminal secretion signal sequence of the α-lectin subunit Aga2p to be modified from Versicolor Versicolor The laccase gene LAC3 gene sequence was successfully anchored on the surface of the cell wall of Saccharomyces cerevisiae, and finally a new whole-cell catalyst was obtained. Compared with the control strain, this strain can still reach 90% of the original enzyme activity after being placed at room temperature for 25 days. %, and in a wastewater environment, its degradation rates for bisphenol A and SMZ reach 60% and 50%, respectively. In the bacterial surface display system, a variety of carrier proteins secreted by Gram-negative bacteria have broad application prospects. Among them, ice crystal nucleoprotein is an outer membrane protein secreted by more than ten kinds of Gram-negative bacteria such as Pseudomonas syringae, Erwinia, Xanthomonas, etc., and is usually located on the cell surface. The protein has a rapid secretion function, which can quickly form ice crystals in pure water at -2°C to -4°C. In general, the protein is composed of three domains: N-terminal, C-terminal, and middle repeat unit. These three domains account for 15%, 4%, and 81% of the entire sequence, respectively. The researchers found that the N-terminal domain is involved in the localization and transmembrane transportation of INP on the surface of microbial cells, while the C-terminal domain is related to the secretion and transportation of INP. The intermediate repeat domain is rich in a variety of hydrophilic amino acids, which can be Form a symmetrical conformation such as β-sheet, which makes INP thermodynamically stable. Jung et al. found that the structure of the intermediate repeat unit can be modified according to experimental purposes without affecting the function of the fusion protein. In addition to the full sequence of INP, only the C-terminal or N-terminal domain of INP can be successfully applied to microbial cell surface display. system. At present, the special structure of INP makes it used to display a variety of foreign proteins and has good application prospects in the fields of antibiotic degradation, heavy metal adsorption and industrial enzyme display.

Passenger protein is also called target protein. At present, more and more researchers use cell surface display technology to fuse passenger protein with a variety of carrier proteins to obtain whole-cell biocatalysts with special functions. Affected by the size and folding of the passenger protein's own group, its secretion and transport in the periplasm will also be disturbed to a certain extent. Therefore, in the actual application process, the choice of passenger protein is very important. At present, in the field of removing environmental pollutants, researchers divide passenger proteins into two categories; one is the target protein naturally secreted by microorganisms such as fungi and bacteria, and this type of target protein contains some organic matter-degrading enzymes such as white protein. The laccases LACC6 (GenBank accession No.KX815352), LACC9 (accession No. KX815353) and LACC10 (accession No.KX815354) of saprophytes have been proven to rapidly degrade chlorophenols, nitrophenols and sulfonamide antibiotics. An organic pollutant; the other is a synthetic polypeptide or short peptide, which can be correctly expressed on the surface of the host microorganism or on the periplasm of the host microorganism after being modified by molecular biology methods. Liu et al. successfully expressed the heptapeptide containing lysine and cysteine ​​on the outer membrane surface of E. coli, which can specifically adsorb Hg2+ and MeHg95 in the carp.

Microscopic imaging analysis to detect whether the INP fusion protein is successfully anchored on the surface of the expression strain EcN

In order to preliminarily detect whether the INP fusion protein is successfully anchored on the surface of the expression strain EcN, this study uses the reporter protein GFP as a template to design primers Gf and Gr, and connect them to the end of INP by overlap PCR. A recombinant plasmid containing the INP-GFP fusion protein Named pSB18A/INP-N-LG, after digestion, ligation, transformation and screening, the Escherichia coli containing plasmid pSB18A/INP-N-LG was obtained and named EcN-ILG. EcN-ILG was inoculated into 5 mL of LB test tube culture medium, and Ampicillin at a final concentration of 50 μg/mL was added to the culture medium. After culturing for 6 hours at 37°C and 180 rpm, the bacteria were collected by centrifugation (8000 rpm) and washed twice with PBS (pH=7.0). Then the expression of GFP was verified in two parts: one was to add 10 μL bacterial hanging drop. On a glass slide, use a fluorescent microscope (LeiKa, Germany) to perform bright field observation (eyepiece × objective lens = 10 × 40), and after finding a suitable field of view, turn on the excitation light and switch the filter to blue light to generate green fluorescence and observe the display. Micro imaging; instead, take 250 μL of the remaining bacterial suspension on a 96-well plate, set up multiple controls, and use a microplate reader to detect the protein intensity of GFP in the range of 485-515 nm.