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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

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.

In order to preliminarily detect whether the INP fusion protein was successfully anchored on the outer membrane of the expression strain, this study used overlap PCR amplification method to connect the gfp gene sequence to the end of the INP fusion protein. It can be directly observed that the recombinant engineered strain EcN-ILG emits green fluorescence through fluorescence microscope imaging, while the control strain EcN-PSB18a cannot emit green fluorescence. This result preliminarily indicates that the INP fusion protein is anchored on the surface of EcN cells.

Analysis of subcellular fractionation and detection of INP fusion protein expression on cell surface

The recombinant engineered strain EcN-IL and the control strain were inoculated into 100 mL LB medium containing Ampicillin at an inoculum of 1%, cultured at 37°C to OD600=1.5, and subjected to two ultra-high-speed centrifugation according to the method described by Shi et al. (4 ℃, 39000 rpm, 1 h) after collecting cell lysate (T), soluble fraction (S), outer membrane fraction (OM), inner membrane fraction (IM). Subsequently, an equal volume of SDS gel loading buffer was added, and the inactivated components were collected after 5 minutes in a boiling water bath at 98°C. The expression of the fusion protein in bacterial cells was detected by 15% polyacrylamide gel electroporation and western blotting. Location. The western blot was performed with reference to the method reported by Wei et al. (et al., 2014). This experiment uses His-tag as the primary antibody and goat anti-mouse IgG as the secondary antibody.

Cell fractionation was used to analyze the subcellular localization of INP fusion protein expression. Figure 2-3a shows that the INP fusion protein was successfully anchored on the outer cell membrane of the expressing strain EcN after SDS-PAGE. The protein size is 104 KDa , And no band of fusion protein was detected in the inner membrane component of EcN. Figure 2-3a. After western blotting, it is found that the total bacterial protein lysate (T) and the outer cell membrane fraction (OM) obtained by centrifugation contain fusion protein, while the inner cell membrane fraction (IM) is present. No obvious bands were found in the periplasmic component (S). The experiment found that certain bands also appeared in the periplasmic component (S). Therefore, it is speculated that the INP fusion protein may be synthesized in the body first, and then anchored on the surface of the cell membrane. During the experiment, EcN carrying empty pSB18A was used as a control, and it was found that there was no expression of fusion protein in the cytoplasm, inner cell membrane, and outer cell membrane components.

Contribution: LZU-HS-CHINA 2021

Functional verification and reduction rate comparison of three selenite-reduced proteins

In this study, the whole genome of LZ-01 was compared with the previously reported selenite reductase in the NCBI database, and three proteins with a similarity of 28-32% were obtained. The coding genes are SAV 0956, SAKG03 26900, and SAKOR 01018, respectively. See Table 2-1. Three genes were obtained by PCR from LZ-01, as shown in Figure 2-2. After that, the three genes were constructed on the pET 28a(+) inducible vector by the method of molecular cloning, and they were verified in vitro after purification by a nickel column. The verification results can be seen from Figure 2-3a, SAV 0956 gene expression has the strongest protein reduction ability, which reduced 59.73% Se (IV) after 0.5 h of reaction, followed by SAKOR 01018 which reduced 29.2% Se (IV). ), and SAKG03 26900 has almost no reduction ability. Therefore, we took the SAV0956 gene as the research object, and named the selenite-reduced protein expressed by it as SerV01. As shown in Figure 2-3b, the reaction centrifuge tubes from left to right are: control (no protein), the proteins expressed by SAKG03 26900, SAKOR, 01018SAV 0956, it can be observed that the control group has no red material at all, and the reduction gives the most red material Is the protein expressed by the SAV0956 gene.

Table 2-1 Results of gene comparison

Figure 2-2 PCR results of the three genes.

Fig2-3 The Se (IV) reduction rate of SAV0956, SAKG0326900, SAKQR01018with 1 mM Se (IV) in Tris-HCl 7.4.

The previously reported selenite reductase to verify the reducing ability has been identified, glutathione reductase in Escherichia coli, dehydrogenase I in Clostridium, and nitrite reduction in Serratia Enzymes, the dissimilation enzyme Srr in Bacillus selenide, and the NADH:flavin oxidoreductase of rhizobia are thought to be related to the reduction of Se(IV) in aerobic bacteria. The Serratia strain HCNT1 mutant lacking nitrite reductase grows poorly in the presence of 5 mM selenite, and fails to grow in the presence of 25 or 50 mM selenite, increasing selenite sensitivity . The selenite reductase CsrF in Alishewanella sp. WH16-1, the mutant constructed by the suicide allelic recombination method, showed the slow reduction of Se(IV). In Shewanella oneidensis MR-1, the reductase FccA was mutated to reduce selenite by 60%. Most studies have done knockout verification in bacteria, but there are few in vitro verification experiments after purification. Compared with other selenite reductases that have been reported, the enzyme used in this experiment was directly purified and verified in vitro. SerV01 enzyme alone can reduce selenite without the need for multiple enzymes, which is cost-saving and stable. With high performance, it is necessary to explore the applicability of its enzyme activity and reaction conditions.

SerV01 protein Se (IV) reduction enzyme activity dynamics analysis of Michaelis equation and determination of cofactor FMN

Exploring the reaction conditions of the enzyme, it can be seen from Figure 2-4 that when NADH is added as an electron donor, there is a significant decrease in absorbance value from 0 to 150s under the real-time wavelength detection system of U-3900H instrument, indicating the reduction of selenite Enzymes can be combined with electron donors to achieve a reduction effect. There was no change in absorbance in the group without added protein, indicating that NADH was not consumed.

Figure 2-4 Changes in absorbance values for whether NADH is bound or not.

SerV01 protein is similar to the old yellow enzyme family, we further study its enzyme activity kinetics in order to explore the enzyme activity. It can be seen from Figure 2-5 that the Vmax value is 1.119 mol NADH mol-1 s-1 and the Km value is 15.5 μmol/L. Compared with other reductases, the in vitro Se(IV) reduction Vmax of TrxR enzyme in Bacillus Y3 is 12.23 μmol/L, 11.20 μmM min-1 mg-1. For the Srr in Bacillus mLS10, the Km of the enzyme is 145 ± 53 μmol/L, the Vmax is 23 ± 2.5 μM min -1, and the Kcat is 23 ± 2.68 s -1. Enzyme kinetics showed that the low Km value of the enzyme indicates that the enzyme has a high binding capacity to the substrate. Therefore, the selenite reductase SerV01 can be regarded as an effective enzyme for the production of SeNPs.

Figure 2-5 Enzyme activity kinetic curves.

Due to previous reports of NADH flavin oxidoreductase in Rhizobium selenitireducens, dehydrogenase I (Hydrogenase I), sulfite reductase and glutathione peroxidase in Clostridium pasteurianum are all sub- Selenate reductase is used for comparison with the sequence of the target protein SerV01. The Mega 7.0 software was further used to compare SerV01 with protein products of different genera. The results showed that this protein belongs to NADH-dependent flavin redox protein, which is similar to rhizobia selenitireducens NADH: flavin oxidoreductase, and is similar to the known flavin oxidoreductase. Traditional sulfite reductase is similar to selenite reductase from different sources, as shown in Figure 2-6.

Figure 2-6 SerV01 system evolutionary tree

The SerV01 protein structure predicted by the 3DLigandSite server is shown in Figure 2-7. The gray part is the protein backbone, the blue part is the predicted ligand binding site, and the green part is the predicted ligand FMN. As shown in the figure, the ligand is in The protein binds in the pocket and transfers electrons through NAD(P)H to achieve the function of reducing tetravalent selenium to zero valent selenium.

Figure 2-7 Protein structure and ligand mockups.

As the ligand is speculated to be FMN, high performance liquid chromatography was used for verification. The FMN standard sample was prepared as a control, and the prepared ligand solution of SerV01 protein was used as the experimental group to obtain the corresponding absorption peaks, as shown in Figure 2. -8. After measurement, it was found that the peak was separated at 3.05 at the same time, thus proving that the ligand was indeed FMN. The previously reported CsrF selenium-chromium co-reductase also uses FMN as a ligand. The ChrT gene encodes a chromate reductase, also known as flavin mononucleotide (FMN) reductase, which can catalyze the reduction of Cr (VI). ChrR in is also a hexavalent chromium reductase with FMN as a ligand. FMN has become a common ligand for redox proteins in bacteria because the partial dehydrogenation and hydrogenation reaction of isooxazine is a reversible process that participates in the redox reaction of enzymes.

Figure 2-8 Peak time graph of ligands determined by HPLC