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.     

Cell surface display of Chi92 on Escherichia coli using ice nucleation protein for improved catalytic and antifungal activity

The gene encoding chitinase 92 (Chi92) from Aeromonas hydrophila JP10 has been displayed on the cell surface of Escherichia coli using the N-terminal region of ice nucleation proteins (INPN) as an anchoring motif. Immunofluorescence microscopy confirmed that Chi92 was anchored on the cell surface. Western blot analysis further identified the synthesis of INP derivatives containing the N-terminal domain INPN-Chi92 fusion protein of the expected size (112 kDa). Whole cell enzyme assay indicated that the displayed Chi92 showed enhanced catalytic activity toward colloidal chitin. In addition, the Chi92-displayed cells exhibited inhibitory effects on the mycelial growth of phytopathogenic fungi, including Fusarium decemcellulare, Sclerotium rolfsii, Rhizoctonia solani kuhn, and Fusarium oxysporum f.sp. melonis. This study suggested that the INP-based display systems can be used to express a large protein (90 kDa Chi92) on the cell surface of E. coli without growth inhibition. In addition, the display of chitinase on the cell surface may provide an attractive method for the development of biocontrol agents against phytopathogenic fungi.     

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.     

冰晶核蛋白及其在细菌表面展示技术中的应用

冰晶核蛋白(ice nucleation protein,INP)是一种分泌型外膜蛋白,广泛分布于丁香假单胞菌,荧光假单胞菌和其他革兰氏阴性菌中。由于其在相对高温下(-2~-4℃)形成冰核的特性,INP最早应用于生物制冷领域。在细菌表面展示技术中,冰晶核蛋白作为运载蛋白得到广泛的应用。与其他的表面技术载体蛋白相比较,冰晶核蛋白具有稳定表达外源蛋白及展示分子量较大的外源蛋白的优点。INP细胞表面展示技术已被应用于全细胞生物催化剂、全细胞吸附剂和环境污染物降解剂等的开发,本文将简述INP表面展示技术的研究进展。     

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.     

Cell surface display of carbonic anhydrase on Escherichia coli using ice nucleation protein for CO₂ sequestration

Carbonic anhydrase (CA) has recently gained renewed interests for its potential as a mass-transfer facilitator for CO(2) sequestration. However, the low stability and high price severely limit its applications. In this work, the expression of α-CA from Helicobacter pylori on the outer membrane of Escherichia coli using a surface-anchoring system derived from ice nucleation protein (INP) from Pseudomonas syringae was developed. To find the best surface anchoring motif, full-length INP (114 kDa), truncated INP (INP-NC, 33 kDa), and INP's N-domain with first two subunits (INP-N, 22 kDa) were evaluated. Two vectors, pKK223-3 and pET22b(+), with different promoters (T7 and Tac) were used to construct the fusion genes, and for each vector, three recombinant strains, each expressing a different length of the fusion protein, were obtained. SDS-PAGE, Western blot, immunofluorescence microscopy, FACS, and whole-cell ELISA confirmed the expression of fusion proteins on the surface of E. coli. The smallest fusion protein with INP-N as the anchoring motif had the highest expression level and CA activity, suggesting that INP-N is the best carrying protein due to its smaller size. Also, the T7 promoter in pET22b(+) induced with 0.2 mM IPTG gave high protein expression levels, whereas the Tac promoter in pKK223-3 gave low expression levels. The surface displayed CA was at least twofold more stable than that of the free form, and did not show any adverse effect on cell growth and outer membrane integrity. Cells with surface displayed CA were successfully used to facilitate CO(2) sequestration in contained liquid membrane (CLM).     

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     

Bacterial Cell Surface Display of an Enzyme Library for Selective Screening of Improved Cellulase Variants

The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.     

Bacterial cell surface display of an enzyme library for selective screening of improved cellulase variants

The bacterial surface display method was used to selectively screen for improved variants of carboxymethyl cellulase (CMCase). A library of mutated CMCase genes generated by DNA shuffling was fused to the ice nucleation protein (Inp) gene so that the resulting fusion proteins would be displayed on the bacterial cell surface. Some cells displaying mutant proteins grew more rapidly on carboxymethyl cellulose plates than controls, forming heterogeneous colonies. In contrast, cells displaying the nonmutated parent CMCase formed uniform tiny colonies. These variations in growth rate were assumed to result from altered availability of glucose caused by differences in the activity of variant CMCases at the cell surface. Staining assays indicate that large, rapidly growing colonies have increased CMCase activity. Increased CMCase activity was confirmed by assaying the specific activities of cell extracts after the expression of unfused forms of the variant genes in the cytoplasm. The best-evolved CMCases showed about a 5- and 2.2-fold increase in activity in the fused and free forms, respectively. Sequencing of nine evolved CMCase variant genes showed that most amino acid substitutions occurred within the catalytic domain of the enzyme. These results demonstrate that the bacterial surface display of enzyme libraries provides a direct way to correlate evolved enzyme activity with cell growth rates. This technique will provide a useful technology platform for directed evolution and high-throughput screening of industrial enzymes, including hydrolases.     

Expression of single chain antibodies (ScFvs) for c-myc oncoprotein in recombinant Escherichia coli membranes by using the ice-nucleation protein of Pseudomonas syringae

The ice nucleation protein (INP) is a glycosyl phosphatidylinositol anchored outer membrane protein found in certain Gram-negative bacteria. In this study, the INP from Pseudomonas syringae was applied as a fusion partner with the single chain antibody fragment (ScFv) against the human oncoprotein c-myc. Two new plasmids pNinaZ-myc and pNinaZScFv-BsaA1 were constructed and cloned into Escherichia coli JM109. The expression of the fusion protein was successfully demonstrated in the cloned cells. The fusion proteins had no effect on the viability of the host cells. Ice nucleation activity measurements and flow cytometry studies were followed to investigate the membrane expression of the fusion protein.     

Surface Display of GFP by <Emphasis Type="Italic">Pseudomonas Syringae</Emphasis> Truncated Ice Nucleation Protein in Attenuated <Emphasis Type="Italic">Vibrio Anguillarum</Emphasis> Strain

Microbial cell surface display of foreign proteins has been widely developed for many potential applications in live vaccine construction, whole-cell biocatalysts, and bioadsorption. To investigate the feasibility of displaying heterologous proteins on the surface of attenuated Vibrio anguillarum strain for potential multivalent live vaccine development, different display systems were built upon a truncated ice nucleation protein (INP) from Pseudomonas syringae ICMP3023 whose N- and C-terminal domains were considered to be the putative membrane-anchoring motifs. Green fluorescent protein (GFP), as a reporter, was fused with the display systems in different forms of N-GFP, NC-GFP, and N-GFP-C. Analysis of the total expression level and surface localization of GFP demonstrated that the truncated P. syringae INP could be used to display foreign protein in V. anguillarum, while the system of N-GFP showed the higher levels of total expression and surface display based on unit cell density among the three and might be available for further carrier vaccine development.     

Decorating microbes: surface display of proteins on Escherichia coli

Bacterial surface display entails the presentation of recombinant proteins or peptides on the surface of bacterial cells. Escherichia coli is the most frequently used bacterial host for surface display and, as such, a variety of E. coli display systems have been described that primarily promote the surface exposure of peptides and small proteins. By contrast, display systems based on autotransporter proteins (ATs) and ice nucleation protein (INP) are excellent systems for the display of large and complex proteins, and are therefore of considerable biotechnological relevance. Here, we review recent advances in AT and INP-mediated display and their biotechnological applications. Additionally, we discuss several promising alternative display methods, as well as novel bacterial host organisms.     

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.     

Expression and surface display of Cellulomonas endoglucanase in the ethanologenic bacterium Zymobacter palmae

In order to reduce the cost of bioethanol production from lignocellulosic biomass, we developed a tool for cell surface display of cellulolytic enzymes on the ethanologenic bacterium Zymobacter palmae. Z. palmae is a novel ethanol-fermenting bacterium capable of utilizing a broad range of sugar substrates, but not cellulose. Therefore, to express and display heterologous cellulolytic enzymes on the Z. palmae cell surface, we utilized the cell-surface display motif of the Pseudomonas ice nucleation protein Ina. The gene encoding Ina from Pseudomonas syringae IFO3310 was cloned, and its product was comprised of three functional domains: an N-terminal domain, a central domain with repeated amino acid residues, and a C-terminal domain. The N-terminal domain of Ina was shown to function as the anchoring motif for a green fluorescence protein fusion protein in Escherichia coli. To express a heterologous cellulolytic enzyme extracellularly in Z. palmae, we fused the N-terminal coding sequence of Ina to the coding sequence of an N-terminal-truncated Cellulomonas endoglucanase. Z. palmae cells carrying the fusion endoglucanase gene were shown to degrade carboxymethyl cellulose. Although a portion of the expressed fusion endoglucanase was released from Z. palmae cells into the culture broth, we confirmed the display of the protein on the cell surface by immunofluorescence microscopy. The results indicate that the N-terminal anchoring motif of Ina from P. syringae enabled the translocation and display of the heterologous cellulase on the cell surface of Z. palmae.     

Cell surface display of poliovirus receptor on Escherichia coli, a novel method for concentrating viral particles in water

The lack of efficient methods for concentrating viruses in water samples leads to underreporting of viral contamination in source water. A novel strategy for viral concentration was developed using the expression of target virus receptors on bacterial cells. Poliovirus type 1, the most studied enterovirus, was used as a surrogate for enteric viruses. The human poliovirus receptor (hPVR) gene was expressed on the surface of Escherichia coli cells by using the ice nucleation protein (INP) gene. The hPVR gene was ligated to the 3' end of the INP gene after the removal of the stop codon. The resulting open reading frame (ORF) was used for the projection of hPVR onto the outer membrane of E. coli. Gene expression was tested by SDS-PAGE, Western blot, and dot blot analyses, and virion capture ability was confirmed by transmission electron microscopy. The application of engineered E. coli cells for capturing viruses in 1-liter samples of source and drinking water resulted in 75 to 99% procedural recovery efficiency. Cell surface display of viral receptors on bacterial cells opens a new prospect for an efficient and inexpensive alternative tool for capturing and concentrating waterborne viruses in water samples.     

The yeast inositol polyphosphate 5-phosphatase Inp54p localizes to the endoplasmic reticulum via a C-terminal hydrophobic anchoring tail: regulation of secretion from the endoplasmic reticulum

The budding yeast Saccharomyces cerevisiae has four inositol polyphosphate 5-phosphatase (5-phosphatase) genes, INP51, INP52, INP53, and INP54, all of which hydrolyze phosphatidylinositol (4,5)-bisphosphate. INP54 encodes a protein of 44 kDa which consists of a 5-phosphatase domain and a C-terminal leucine-rich tail, but lacks the N-terminal SacI domain and proline-rich region found in the other three yeast 5-phosphatases. We report that Inp54p belongs to the family of tail-anchored proteins and is localized to the endoplasmic reticulum via a C-terminal hydrophobic tail. The hydrophobic tail comprises the last 13 amino acids of the protein and is sufficient to target green fluorescent protein to the endoplasmic reticulum. Protease protection assays demonstrated that the N terminus of Inp54p is oriented toward the cytoplasm of the cell, with the C terminus of the protein also exposed to the cytosol. Null mutation of INP54 resulted in a 2-fold increase in secretion of a reporter protein, compared with wild-type yeast or cells deleted for any of the SacI domain-containing 5-phosphatases. We propose that Inp54p plays a role in regulating secretion, possibly by modulating the levels of phosphatidylinositol (4,5)-bisphosphate on the cytoplasmic surface of the endoplasmic reticulum membrane.     

Cloning and expression of afpA, a gene encoding an antifreeze protein from the arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2

The Arctic plant growth-promoting rhizobacterium Pseudomonas putida GR12-2 secretes an antifreeze protein (AFP) that promotes survival at subzero temperatures. The AFP is unusual in that it also exhibits a low level of ice nucleation activity. A DNA fragment with an open reading frame encoding 473 amino acids was cloned by PCR and inverse PCR using primers designed from partial amino acid sequences of the isolated AFP. The predicted gene product, AfpA, had a molecular mass of 47.3 kDa, a pI of 3.51, and no previously known function. Although AfpA is a secreted protein, it lacked an N-terminal signal peptide and was shown by sequence analysis to have two possible secretion systems: a hemolysin-like, calcium-binding secretion domain and a type V autotransporter domain found in gram-negative bacteria. Expression of afpA in Escherichia coli yielded an intracellular 72-kDa protein modified with both sugars and lipids that exhibited lower levels of antifreeze and ice nucleation activities than the native protein. The 164-kDa AFP previously purified from P. putida GR12-2 was a lipoglycoprotein, and the carbohydrate was required for ice nucleation activity. Therefore, the recombinant protein may not have been properly posttranslationally modified. The AfpA sequence was most similar to cell wall-associated proteins and less similar to ice nucleation proteins (INPs). Hydropathy plots revealed that the amino acid sequence of AfpA was more hydrophobic than those of the INPs in the domain that forms the ice template, thus suggesting that AFPs and INPs interact differently with ice. To our knowledge, this is the first gene encoding a protein with both antifreeze and ice nucleation activities to be isolated and characterized.     

Production, secretion, and cell surface display of recombinant Sporosarcina ureae S-layer fusion proteins in Bacillus megaterium

Monomolecular crystalline bacterial cell surface layers (S-layers) have broad application potential in nanobiotechnology due to their ability to generate functional supramolecular structures. Here, we report that Bacillus megaterium is an excellent host organism for the heterologous expression and efficient secretion of hemagglutinin (HA) epitope-tagged versions of the S-layer protein SslA from Sporosarcina ureae ATCC 13881. Three chimeric proteins were constructed, comprising the precursor, C-terminally truncated, and N- and C-terminally truncated forms of the S-layer SslA protein tagged with the human influenza hemagglutinin epitope. For secretion of fusion proteins, the open reading frames were cloned into the Escherichia coli-Bacillus megaterium shuttle vector pHIS1525. After transformation of the respective plasmids into Bacillus megaterium protoplasts, the recombinant genes were successfully expressed and the proteins were secreted into the growth medium. The isolated S-layer proteins are able to assemble in vitro into highly ordered, crystalline, sheetlike structures with the fused HA tag accessible to antibody. We further show by fluorescent labeling that the secreted S-layer fusion proteins are also clustered on the cell envelope of Bacillus megaterium, indicating that the cell surface can serve in vivo as a nucleation point for crystallization. Thus, this system can be used as a display system that allows the dense and periodic presentation of S-layer proteins or the fused tags.     

Anchorage of GFP fusion on the cell surface of <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Here we report the cell surface display of organophosphorus hydrolase (OPH) and green fluorescent protein (GFP) fusion by employing the N- and C-terminal domains of ice nucleation protein (INPNC) as an anchoring motif. An E. coli–Pseudomonas shuttle vector, pNOG33, coding for INPNC–OPH–GFP was constructed for targeting the fusion onto the cell surface of p-nitrophenol (PNP)-degrading P. putida JS444. The surface localization of INPNC–OPH–GFP was verified by cell fractionation, Western blot, proteinase accessibility, and immunofluorescence microscopy. Furthermore, the functionality of the surface-exposed OPH–GFP was demonstrated by OPH assays and fluorescence measurements. Surface display of macromolecular OPH–GFP fusion (63 kDa) neither inhibited cell growth nor affected cell viability. These results suggest that INP is an useful tool for the presentation of heterologous proteins on cell surfaces of indigenous microbes. The engineered P. putida JS444 degraded organophosphates (OPs) as well as PNP rapidly and could be easily monitored by fluorescence. Parathion (100 mg kg⁻¹) could be degraded completely within 15 days in soil inoculated with the engineered strain. These merits make this engineered strain an ideal biocatalyst for in situ bioremediation of OP-contaminated soil.     

An ice nucleation protein from Fusarium acuminatum: cloning,expression, biochemical characterization and computational modeling

Ice nucleation proteins (INP) are a major cause of frost damage in plants and crops. Here, an INP gene from Fusarium acuminatum was optimized, synthesized, expressed in E.coli and subsequently purified and characterized. The protein belongs to the second class of ice nucleation proteins with an optimum pH 5.5, relative activity and stability between pH 5 and 9.5 and up to 45 °C. The protein was fully active and stable in the presence of dimethyl sulfoxide (DMSO), dioxane, acetone and ethyl acetate. Moreover, it retained over 50 % of its original activity in the presence of polyvinyl alcohol. The 3D structure model of the INP-F indicated the protein had three distinct domains as exist in other ice nucleation proteins with some variations. Considering these promising results, INP-F could be a novel candidate for industrial applications.