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surface expression inp

Baozhen Zhang, Lunbo Tan, Yan Yu, Baobei Wang, Zhilong Chen, Jinyu Han, Mengxia Li, Jie Chen, Tianxia Xiao, Balamurali K Ambati, Lintao Cai, Qing Yang, Nihar R Nayak, Jian Zhang, Xiujun Fan
Rationale: The availability of therapeutics to treat pregnancy complications is severely lacking, mainly due to the risk of harm to the fetus. In placental malaria, Plasmodium falciparum- infected erythrocytes (IEs) accumulate in the placenta by adhering to chondroitin sulfate A (CSA) on the surfaces of trophoblasts. Based on this principle, we have developed a method for targeted delivery of payloads to the placenta using a synthetic placental CSA-binding peptide (plCSA-BP) derived from VAR2CSA, a CSA-binding protein expressed on IEs...
2018: Theranostics
Daehwan Kim, Seockmo Ku
One of the main challenges of using recombinant enzymes is that they are derived from genetically-modified microorganisms commonly located in the intracellular region. The use of these recombinant enzymes for commercial purposes requires the additional processes of cell disruption and purification, which may result in enzyme loss, denaturation, and increased total production cost. In this study, the cellulase gene of Bacillus licheniformis ATCC 14580 was cloned, over-expressed, and surface displayed in recombinant Escherichia coli using an ice-nucleation protein (INP)...
February 24, 2018: Molecules: a Journal of Synthetic Chemistry and Natural Product Chemistry
Chao Yang, Xiaoqing Xu, Yanping Liu, Hong Jiang, Yunbo Wu, Ping Xu, Ruihua Liu
Many sites are often co-contaminated with multiple pesticides. To date, there are no reports on simultaneous degradation of different classes of pesticides by a natural microorganism. In this work, we aim at constructing a live biocatalyst able to simultaneously hydrolyze carbaryl and chlorpyrifos. For this purpose, carbaryl hydrolase (CH) was displayed on the cell surface of a chlorpyrifos-degrading bacterium Stenotrophomonas sp. strain YC-1 using N- and C-terminal domain of ice nucleation protein (INPNC) from Pseudomonas syringae INA5 as an anchoring motif...
October 17, 2017: Scientific Reports
Sonia Del Prete, Rosa Perfetto, Mosè Rossi, Fatmah A S Alasmary, Sameh M Osman, Zeid AlOthman, Claudiu T Supuran, Clemente Capasso
The carbonic anhydrase superfamily (CA, EC of metalloenzymes is present in all three domains of life (Eubacteria, Archaea, and Eukarya), being an interesting example of convergent/divergent evolution, with its seven families (α-, β-, γ-, δ-, ζ-, η-, and θ-CAs) described so far. CAs catalyse the simple, but physiologically crucial reaction of carbon dioxide hydration to bicarbonate and protons. Recently, our groups characterised the α-CA from the thermophilic bacterium, Sulfurihydrogenibium yellowstonense finding a very high catalytic activity for the CO2 hydration reaction (kcat = 9...
December 2017: Journal of Enzyme Inhibition and Medicinal Chemistry
Aihua Liu, Ruirui Feng, Bo Liang
In the present work, NAD(+)-dependent formate dehydrogenase (FDH), encoded by fdh gene from Candida boidinii was successfully displayed on Escherichia coli cell surface using ice nucleation protein (INP) from Pseudomonas borealis DL7 as an anchoring protein. Localization of matlose binding protein (MBP)-INP-FDH fusion protein on the E. coli cell surface was characterized by SDS-PAGE and enzymatic activity assay. FDH activity was monitored through the oxidation of formate catalyzed by cell-surface-displayed FDH with its cofactor NAD(+), and the production of NADH can be detected spectrometrically at 340nm...
September 2016: Enzyme and Microbial Technology
Matthew McCallum, Stephanie Tammam, Dustin J Little, Howard Robinson, Jason Koo, Megha Shah, Charles Calmettes, Trevor F Moraes, Lori L Burrows, P Lynne Howell
Pseudomonas aeruginosa is an opportunistic bacterial pathogen that expresses type IVa pili. The pilus assembly system, which promotes surface-associated twitching motility and virulence, is composed of inner and outer membrane subcomplexes, connected by an alignment subcomplex composed of PilMNOP. PilM binds to the N terminus of PilN, and we hypothesize that this interaction causes functionally significant structural changes in PilM. To characterize this interaction, we determined the crystal structures of PilM and a PilM chimera where PilM was fused to the first 12 residues of PilN (PilM·PilN(1-12))...
May 20, 2016: Journal of Biological Chemistry
Bo Liang, Shu Zhang, Qiaolin Lang, Jianxia Song, Lihui Han, Aihua Liu
A novel L-glutamate biosensor was fabricated using bacteria surface-displayed glutamate dehydrogenase (Gldh-bacteria). Here the cofactor NADP(+)-specific dependent Gldh was expressed on the surface of Escherichia coli using N-terminal region of ice nucleation protein (INP) as the anchoring motif. The cell fractionation assay and SDS-PAGE analysis indicated that the majority of INP-Gldh fusion proteins were located on the surface of cells. The biosensor was fabricated by successively casting polyethyleneimine (PEI)-dispersed multi-walled carbon nanotubes (MWNTs), Gldh-bacteria and Nafion onto the glassy carbon electrode (Nafion/Gldh-bacteria/PEI-MWNTs/GCE)...
July 16, 2015: Analytica Chimica Acta
Song Tang, Vinay Allagadda, Hicham Chibli, Jay L Nadeau, Gregory D Mayer
Recent advances in the ability to manufacture and manipulate materials at the nanometer scale have led to increased production and use of many types of nanoparticles. Quantum dots (QDs) are small, fluorescent nanoparticles composed of a core of semiconductor material (e.g. cadmium selenide, zinc sulfide) and shells or dopants of other elements. Particle core composition, size, shell, and surface chemistry have all been found to influence toxicity in cells. The aim of this study was to compare the toxicities of ionic cadmium (Cd) and zinc (Zn) and Cd- and Zn-containing QDs in zebrafish liver cells (ZFL)...
October 2013: Metallomics: Integrated Biometal Science
Nour Karra, Taher Nassar, Alina Nemirovski Ripin, Ouri Schwob, Jürgen Borlak, Simon Benita
Aberrant signaling of the epidermal growth factor receptor (EGFR) is common to a variety of human cancers and is also found to be over-expressed in most cases of non-small cell lung cancer. For the development of a molecularly targeted therapy, cetuximab-conjugated nanoparticles (immunonanoparticles, INPs) are designed and loaded with the lipophilic paclitaxel palmitate (pcpl) prodrug. Oleyl cysteineamide (OCA) is synthesized whereby its amphiphilic nature enables interfacial anchoring and thiol surface functionalization of PLGA NPs, facilitating bioconjugation to cetuximab by thioether bonds...
December 20, 2013: Small
Qiuxiang Zhang, Huili Hou, Ying Lu, Wei Chen, Jin Zhong
OBJECTIVE: The gene of ice nucleation protein (INP), an outer membrane of Pseudomonas syringae was tested to display foreign proteins on the surface of Lactococcus lactis. METHODS: Plasmids pHZ101 and pHZ102 were constructed using gfp (Green Fluorescence Protein) as the reporter gene and N-terminal and NC-terminal of inp as the anchoring units. Plasmids pHZ101 and pHZ102 were subsequently transformed into Escherichia coli JM109 and Lactococcus lactis MG1363. RESULTS: Fluorescence microscope shows that green fluorescence was observed in both recombinant E...
April 4, 2013: Wei Sheng Wu Xue Bao, Acta Microbiologica Sinica
Brian G Rash, Simone Tomasi, H David Lim, Carol Y Suh, Flora M Vaccarino
Gyrification allows an expanded cortex with greater functionality to fit into a smaller cranium. However, the mechanisms of gyrus formation have been elusive. We show that ventricular injection of FGF2 protein at embryonic day 11.5-before neurogenesis and before the formation of intrahemispheric axonal connections-altered the overall size and shape of the cortex and induced the formation of prominent, bilateral gyri and sulci in the rostrolateral neocortex. We show increased tangential growth of the rostral ventricular zone (VZ) but decreased Wnt3a and Lef1 expression in the cortical hem and adjacent hippocampal promordium and consequent impaired growth of the caudal cortical primordium, including the hippocampus...
June 26, 2013: Journal of Neuroscience: the Official Journal of the Society for Neuroscience
Chia-Chi Lin, Tzu-Tsen Liu, Shu-Chen Kan, Chi-Zong Zang, Chiung-Wen Yeh, Jiun-Yan Wu, Jiann-Hwa Chen, Chwen-Jen Shieh, Yung-Chuan Liu
In this study, the cell surface expression system was the first time used to directly produce extracellular enzyme. In the plasmid construction, the truncated ice nucleation protein (INP) was fused with intein (INT) and target protein, D-hydantoinase (DHTase), to form the INP-INT-DHTase gene. The plasmid containing this gene was transformed into Escherichia coli ER2566 cells. The gene construct enables the expression of INP-INT-DHTase fusion protein, which might anchor on cell membrane surface. The induction conditions were studied and optimal conditions were as follows: E...
November 2013: Journal of Bioscience and Bioengineering
Bo Liang, Liang Li, Xiangjiang Tang, Qiaolin Lang, Hongwei Wang, Feng Li, Jianguo Shi, Wei Shen, Ilaria Palchetti, Marco Mascini, Aihua Liu
A genetically engineered Escherichia coli (E. coli) strain displaying glucose dehydrogenase (GDH) with ice-nucleation protein (INP) as the anchoring motif was first constructed. The surface localization and functionality of the fusion protein containing GDH were verified by SDS-PAGE, Western blotting and enzymatic activity assay. The fusion of INP had no effects on the functionality of GDH cofactor binding domain. The activity assay showed that 74.6% of the cell lysate GDH activity was detected in the outer membrane fractions...
July 15, 2013: Biosensors & Bioelectronics
Qianqian Li, Qi Yan, Jinsi Chen, Yan He, Jing Wang, Hongxing Zhang, Ziniu Yu, Lin Li
The ice nucleation protein (INP) of Pseudomonas syringae has gained scientific interest not only because of its pathogenicity of foliar necroses but also for its wide range of potential applications, such as in snow making, frozen food preparation, and surface-display system development. However, studies on the transport activity of INP remain lacking. In the present study, a newly identified INP-gene variant, inaQ, from a P. syringae MB03 strain was cloned. Its structural domains, signal sequences, and the hydrophilicity or hydrophobicity of each domain, were then characterized...
2012: International Journal of Biological Sciences
Mohammed A A Sarhan, Mustaffa Musa, Zainul F Zainuddin
Expressing proteins of interest as fusion to proteins of bacterial envelope is a powerful technique for biotechnological and medical applications. The synthetic gene (VacII) encoding for T-cell epitopes of selected genes of Mycobacterium tuberculosis namely, ESAT6, MTP40, 38 kDa, and MPT64 was fused with N- terminus of Pseudomonas syringae ice nucleation protein (INP) outer membrane protein. The fused genes were cloned into a bacterial expression vector pKK223-3. The recombinant protein was purified by Ni-NAT column...
September 2011: Indian Journal of Experimental Biology
Li-Hai Fan, Ning Liu, Ming-Rui Yu, Shang-Tian Yang, Huan-Lin Chen
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...
December 2011: Biotechnology and Bioengineering
Morteza Abbaszadegan, Absar Alum, Hamed Abbaszadegan, Valerie Stout
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...
August 2011: Applied and Environmental Microbiology
Yulan Yuan, Chao Yang, Cunjiang Song, Hong Jiang, Ashok Mulchandani, Chuanling Qiao
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...
February 2011: Biodegradation
Chao Yang, Roland Freudl, Chuanling Qiao, Ashok Mulchandani
A genetically engineered Escherichia coli strain coexpressing organophosphorus hydrolase (OPH) and methyl parathion hydrolase (MPH) was constructed for the first time by cotransforming two compatible plasmids. Since these two enzymes have different substrate specificities, the coexpression strain showed a broader substrate range than strains expressing either one of the hydrolases. To reduce the mass transport limitation of organophosphates (OPs) across the cell membrane, MPH and OPH were simultaneously translocated to the periplasm and cell surface of E...
January 2010: Applied and Environmental Microbiology
Zhongqin Wu, Lei Qin, Virginia K Walker
Isolates of 'Pseudomonas borealis' were recovered after ice-affinity selection of summer-collected soils. 'P. borealis' DL7 was further characterized and shown to have ice nucleation activity (INA), a property that allows the crystallization of ice at temperatures close to the melting point, effectively preventing the supercooling of water. INA was optimally detected after culturing at temperatures consistent with psychrophilic growth. The sequence encoding the 'P. borealis' ice nucleation protein (INP) was obtained using both PCR and chromosome walking...
April 2009: Microbiology
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