Functional display of foreign protein on surface of Escherichia coli using N-terminal domain of ice nucleation protein

We investigated the ability of the N-terminal domain of InaK, an ice nucleation protein from Pseudomonas syringae KCTC1832, to act as an anchoring motif for the display of foreign proteins on the Escherichia coli cell surface. Total expression level and surface display efficiency of green fluorescent protein (GFP) was compared following their fusion with either the N-terminal domain of InaK (InaK-N), or with the known truncated InaK containing both N- and C-terminal domains (InaK-NC). We report that the InaK-N/GFP fusion protein showed a similar cell surface display efficiency ( approximately 50%) as InaK-NC/GFP, demonstrating that the InaK N-terminal region alone can direct translocation of foreign proteins to the cell surface and can be employed as a potential cell surface display motif. Moreover, InaK-N/GFP showed the highest levels of total expression and surface display based on unit cell density. InaK-N was also successful in directing cell surface display of organophosphorus hydrolase (OPH), confirming its ability to act as a display motif.     

Surface display of transglucosidase on Escherichia coli by using the ice nucleation protein of Xanthomonas campestris and its application in glucosylation of hydroquinone

A surface anchoring motif using the ice nucleation protein (INP) of Xanthomonas campestris pv. campestris BCRC 12,846 for display of transglucosidase has been developed. The transglucosidase gene from Xanthomonas campestris pv. campestris BCRC 12,608 was fused to the truncated ina gene. This truncated INP consisting of N- and C-terminal domains (INPNC) was able to direct the expressed transglucosidase fusion protein to the cell surface of E. coli with apparent high enzymatic activity. The localization of the truncated INPNC-transglucosidase fusion protein was examined by Western blot analysis and immunofluorescence labeling, and by whole-cell enzyme activity in the glucosylation of hydroquinone. The glucosylation reaction was carried out at 40 degrees C for 1 h, which gave 23 g/L of alpha-arbutin, and the molar conversion based on the amount of hydroquinone reached 83%. The use of whole-cells of the wild type strain resulted in an alpha-arbutin concentration of 4 g/L and a molar conversion of 16% only under the same conditions. The results suggested that E. coli displaying transglucosidase using truncated INPNC as an anchoring motif can be employed as a whole-cell biocatalyst in glucosylation.     

Surface display of MPH on Pseudomonas putida JS444 using ice nucleation protein and its application in detoxification of organophosphates

Methyl parathion hydrolase (MPH) has been displayed on the surface of microorganisms for the first time using only N- and C-terminal domains of the ice nucleation protein (INPNC) from Pseudomonas syringae INA5 as an anchoring motif. A shuttle vector pINCM coding for INPNC-MPH was constructed and used to target MPH onto the surface of a natural p-nitrophenol (PNP) degrader, Pseudomonas putida JS444, overcoming the potential substrate uptake limitation. Over 90% of the MPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of the INPNC-MPH fusion was further verified by Western blot analysis and immunofluorescence microscopy. The engineered P. putida JS444 degraded organophosphates as well as PNP rapidly without growth inhibition. Compared to organophosphorus hydrolase-displaying systems reported, changes in substrate specificity highlight an important potential use of the engineered strain for the clean-up of specific organophosphate nerve agents.     

Improved phosphate biosorption by bacterial surface display of phosphate-binding protein utilizing ice nucleation protein

The conventional enhanced biological phosphorus removal (EBPR) system often deteriorates at low chemical oxygen demand (COD) or under aeration conditions. A new approach that incorporates phosphate-eutrophic wastewater remediation was introduced through immobilization of an intracellular phosphate-binding protein (PBP) onto the surface of Pseudomonas putida or Escherichia coli , using the N-terminal anchor (InaQ-N) of a newly identified ice nucleation protein from Pseudomonas syringae . A green fluorescent protein-fusion protein was expressed and used to confirm surface localization. The PBP was then targeted to the surface of E. coli JM109 and P. putida AB92019. The engineered P. putida and E. coli microorganisms were capable of absolute biosorption of total phosphates at rates of 60 and 80 mg L⁻¹, respectively, over 5 h. In the recombinant P. putida cells, a surface-immobilized PBP fusion that had three tandemly repeated InaQ-Ns exhibited the maximum increment in phosphate biosorption, at sixfold compared with the control strain. Even heat-killed recombinant cells of either P. putida or E. coli retained substantial biosorptive activities. The current study demonstrates that the bacterial surface display of PBP should be considered as a strong contender for generating organisms capable of functioning in EBPR systems under low COD, resulting in improved removal of eutrophic phosphorus from wastewaters.     

Cell surface display of CD8 ecto domain on Escherichia coli using ice nucleation protein

A new system for cell surface display of recombinant proteins on Escherichia coli was tested for expression of the ecto domain of CD8, which is the surface protein of human T cytotoxic lymphocytes. Immunofluorescence microscopy, ELISA, and immunodot blotting confirmed successful expression of the CD8 ecto domain fused to ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. © Rapid Science Ltd. 1998     

Cell surface display of organophosphorus hydrolase using ice nucleation protein

A new anchor system based on the ice nucleation protein (InaV) from Pseudomonas syringae INA5 was developed for cell surface display of functional organophosphorus hydrolase (OPH). The activity and stability of cells expressing the truncated InaV (INPNC)-OPH fusions were compared to cells with surface-expressed OPH using two other fusion anchors based on Lpp-OmpA and the truncated InaK protein. Whole cell activity was as much as 5-fold higher using the InaV anchor. Majority of the OPH activity was located on the cell surface as determined by protease accessibility and cell fractionation experiments. The surface localization of OPH was further verified by immunofluorescence microscopy. Constitutive expression of OPH on the surface using the InaV anchor resulted in no cell lysis or growth inhibition, in contrast to the Lpp-OmpA anchor. Suspended cultures also exhibited good stability, retaining almost 100% activity over a period of 3 weeks. Therefore, the InaV anchor system offers an attractive alternative to the currently available surface anchors, providing high-level expression and superior stability.     

利用冰核蛋白N末端结构域在大肠杆菌表面展示粘质沙雷氏菌脂肪酶

【目的】利用细胞表面工程技术将活性脂肪酶展示于大肠杆菌细胞表面并对展示脂肪酶的酶学性质进行研究。【方法】将丁香假单胞菌冰核蛋白N末端结构域序列与粘质沙雷氏菌脂肪酶编码基因融合,构建成脂肪酶表面展示载体,并转化大肠杆菌BL21(DE3)。【结果】重组菌以终浓度0.05 mmol/L异丙基硫代-D-半乳糖苷(IPTG)、25°C条件下诱导培养,16 h后表面展示脂肪酶活力达到最大值1 852 U/g细胞干重。表面展示酶的最适pH为9.0,最适反应温度为40°C,表面展示酶热稳定性较游离酶有较大提高,在40°C孵育1 h后仍能保持90%以上的酶活力。【结论】以上结果表明细菌表面展示技术为脂肪酶固定提供了一个很有前景的替代方法。     

Cell surface display of salmobin, a thrombin-like enzyme from Agkistrodon halys venom on Escherichia coli using ice nucleation protein

Cell surface display on Escherichia coli using ice nucleation protein was performed in order to develop a new expression system for recombinant eukaryotic proteins. Salmobin, the thrombin-like enzyme obtained from Korean snake (Agkistrodon halys) venom was displayed on the surface of Escherichia coli fused to the C-terminus of the ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. The thrombin cleavage site was inserted between salmobin and INP. The presence of salmobin on the bacterial cell surface was verified by SDS-PAGE, Western blotting, whole cell ELISA, and immunofluorescence microscopy. After thrombin cleavage the thrombin-like enzyme activity of recombinant salmobin was tested and verified. We concluded that INP-based cell surface display can be used as a novel expression system for eukaryotic proteins.     

A kinetic model describing cell growth and production of highly active, recombinant ice nucleation protein in Escherichia coli

A structured kinetic model, which describes the production of the recombinant ice nucleation protein in different conditions, was applied. The model parameters were estimated based on the variation of the specific growth rate and the intracellular product concentration during cultivation. The equations employed relate the cellular plasmid content or plasmid copy number with the cloned-gene expression; these correlations were successfully tested on the experimental data. The optimal nutrient conditions for the growth of Escherichia coli expressing the inaZ gene of Pseudomonas syringae were determined for the production of active ice nucleation protein. The kinetics of the cultures expressing the inaZ gene were studied in a bioreactor at different growth temperatures and nutrient conditions.     

Translocation of green fluorescent protein to cyanobacterial periplasm using ice nucleation protein

The translocation of proteins to cyanobacterial cell envelope is made complex by the presence of a highly differentiated membrane system. To investigate the protein translocation in cyanobacterium Synechococcus PCC 7942 using the truncated ice nucleation protein (InpNC) from Pseudomonas syringae KCTC 1832, the green fluorescent protein (GFP) was fused in frame to the carboxyl-terminus of InpNC. The fluorescence of GFP was found almost entirely as a halo in the outer regions of cells which appeared to correspond to the periplasm as demonstrated by confocal laser scanning microscopy, however, GFP was not displayed on the outermost cell surface. Western blotting analysis revealed that InpNC-GFP fusion protein was partially degraded. The N-terminal domain of InpNC may be susceptible to protease attack; the remaining C-terminal domain conjugated with GFP lost the ability to direct translocation across outer membrane and to act as a surface display motif. The fluorescence intensity of cells with periplasmic GFP was approximately 6-fold lower than that of cells with cytoplasmic GFP. The successful translocation of the active GFP to the periplasm may provide a potential means to study the property of cyanobacterial periplasmic substances in response to environmental changes in a non-invasive manner.     

Bacterial cell surface display for epitope mapping of hepatitis C virus core antigen

Cell surface expression of protein has been widely used to display enzymes and antigens. Here we show that Pseudomonas syringae ice nucleation protein with a deletion of internal repeating domain (INC) can be used in Escherichia coli to display peptide in a conformationally active form on the outside of the folded protein by fusing to the C-terminus of INC. Diagnostic potential of this technology was demonstrated by effective mapping of antigenic epitopes derived from hepatitis C virus (HCV) core protein. Amino acids 1-38 and 26-53 of HCV core protein were found to react more sensitively in a native conformation with the HCV patient sera than commercial diagnostic antigen, c22p (amino acids 10-53) by display-ELISA. These results demonstrate that the bacterial cell surface display using INC is useful for peptide presentation and thus epitope mapping of antigen.     

Enhancing the stability of xylanase from Cellulomonas fimi by cell-surface display on Escherichia coli

Aims: The cell‐surface display of Cex, which encodes xylanase and exoglucanase from Cellulomonas fimi, was constructed on Escherichia coli using PgsA as the anchor protein. Characterization of the cell‐surface display of Cex was performed. Methods and Results: PgsA was fused to the N‐terminus of Cex and six histidines were utilized as spacers between the targeting and anchor proteins. Successful cell‐surface display of Cex was demonstrated by Western blot and immunofluorescence analyses on E. coli C41 (DE3). According to the time‐course analysis, the xylanase activity of Cex was achieved at 49 U g^?1 dry cell weight after 12 h culture at 37°C. The optimal temperature and pH ranges of the cell‐surface displayed protein with whole‐cell were broader than the corresponding ranges of the purified form. Further determination of thermostability indicated that the half‐life of cell‐surface displayed Cex was 1·6 times longer than that of purified Cex at 60°C. Conclusions: We have successfully developed the cell‐surface display of xylanase on E. coli. The cell‐surface display can enhance the stability of xylanase against changes in temperature and has the potential of becoming a whole‐cell biocatalyst for industrial applications, such as biobleaching of paper and production of renewable energy. Significance and Impact of the Study: The results demonstrated that the cell‐surface display of xylanase embedded in the cell membrane is more stable than that of the purified enzyme. Thus, to improve the stability of heterologous proteins production, cell‐surface display using the PgsA anchor protein as a tool can be considered in E. coli.     

Cell surface display of hepatitis B virus surface antigen by using Pseudomonas syringae ice nucleation protein

A new system designed for cell surface display of recombinant proteins on Escherichia coli was evaluated for expression of eukaryotic viral antigens. The major surface antigen of hepatitis B virus (HBsAg) was fused to the ice nucleation protein (INP), an outer membrane protein of Pseudomonas syringae. Western blotting, immunofluorescence microscopy, whole-cell ELISA, and ice nucleation activity assay confirmed expression of recombinant proteins on the surface of Escherichia coli. This study indicated that INP-based cell surface display can be used for epitope mapping and recombinant bacteria expressing hepatitis viral antigens may be used for developing live vaccines.     

Cell surface display of synthetic phytochelatins using ice nucleation protein for enhanced heavy metal bioaccumulation.

Synthetic phytochelatins (ECs) composed of (Glu-Cys)nGly are protein analogs of phytochelatin that exhibit improved metal-binding capacity over metallothioneins (MTs). Expression of EC20 on the surface of E. coli using the Lpp-OmpA anchor resulted in improved bioaccumulation of cadmium and mercury, providing a new method for treating heavy metal contamination. To further improve the whole-cell accumulation of heavy metals, EC20 was expressed on the surface of Moraxella sp., a bacterium known to survive in contaminated environments, using the truncated ice nucleation protein (INPNC) anchor. Production of EC20 was approximately three-fold higher in Moraxella sp. than E. coli. As a consequence, the mercury-binding capacity of the recombinant Moraxella sp. was increased by more than 10-fold. Owing to the very high level of surface expression, the use of Moraxella sp. and INPNC anchor may prove to be useful for the remediation of other environmental contaminants.     

Cold requirement for maximal activity of the bacterial ice nucleation protein INAZ in transgenic plants

The bacterial ice nucleation gene inaZ confers production of ice nuclei when transferred into transgenic plants. Conditioning of the transformed plant tissue at temperatures near 0°C greatly increased the ice nucleation activity in plants, and maximum ice nucleation activity was achieved only after low-temperature conditioning for about 48 h. Although the transgenic plants contain similar amounts of inaZ mRNA at both normal and low temperatures, low temperatures are required for accumulation of INAZ protein. We propose that the stability of the INAZ protein and thus ice nucleation activity in the transgenic plants is enhanced by low-temperature conditioning.     

Protein cell surface display in Gram-positive bacteria: from single protein to macromolecular protein structure

In the course of evolution, Gram-positive bacteria, defined here as prokaryotes from the domain Bacteria with a cell envelope composed of one biological membrane (monodermita) and a cell wall composed at least of peptidoglycan and covalently linked teichoic acids, have developed several mechanisms permitting to a cytoplasmic synthesized protein to be present on the bacterial cell surface. Four major types of cell surface displayed proteins are currently recognized: (i) transmembrane proteins, (ii) lipoproteins, (iii) LPXTG-like proteins and (iv) cell wall binding proteins. The subset of proteins exposed on the bacterial cell surface, and thus interacting with extracellular milieu, constitutes the surfaceome. Here, we review exhaustively the current molecular mechanisms involved in protein attachment within the cell envelope of Gram-positive bacteria, from single protein to macromolecular protein structure.     

Yeast surface display of full-length human microtubule-associated protein tau

Microtubule-associated protein tau is an intrinsically disordered, highly soluble protein found primarily in neurons. Under normal conditions, tau regulates the stability of axonal microtubules and intracellular vesicle transport. However, in patients of neurodegeneration such as Alzheimer's disease (AD), tau forms neurofibrillary deposits, which correlates well with the disease progression. Identifying molecular signatures in tau, such as posttranslational modification, truncation, and conformational change has great potential to detect earliest signs of neurodegeneration and develop therapeutic strategies. Here, we show that full-length human tau, including the longest isoform found in the adult brain, can be robustly displayed on the surface of yeast Saccharomyces cerevisiae. Yeast-displayed tau binds to anti-tau antibodies that cover epitopes ranging from the N-terminus to the 4R repeat region. Unlike tau expressed in the yeast cytosol, surface-displayed tau was not phosphorylated at sites found in AD patients (probed by antibodies AT8, AT270, AT180, and PHF-1). However, yeast-displayed tau showed clear binding to paired helical filament (PHF) tau conformation-specific antibodies Alz-50, MC-1, and Tau-2. Although the tau possessed a conformation found in PHFs, oligomerization or aggregation into larger filaments was undetected. Taken together, yeast-displayed tau enables robust measurement of protein interactions and is of particular interest for characterizing conformational change.     

Molecular cloning of an ice nucleation gene from Erwinia ananas and its expression in Escherichia coli

Abstract An approximately 7 kbp genomic DNA fragment was cloned from an ice nucleation-active (ina) strain of Erwinia ananas and defined as to its restriction enzyme site. When the DNA fragment was introduced into E. coli MM294, a potent ice nucleation activity was expressed. Both 0.7 kbp truncation from the 5'-end and 1.7 kbp truncation from the 3'-end were also effective in expressing the ice nucleation activity in E. coli . Therefore, the resulting DNA fragment of approximately 5 kbp was considered to be an ina gene and named ina A. It existed as a unique gene in this strain of E. ananas . No corresponding ina gene existed in an ice nucleation-inactive strain of E. milletiae .