Expression of Carboxymethylcellulase on the Surface of Escherichia Coli Using Pseudomonas Syringae Ice Nucleation Protein

Jung, Heung-Chae; Park, Joon‐Hyun; Park, Seung-Hwan; Lebeault, J. M.; Pan, Jae-Gu

doi:10.1016/s0141-0229(97)00224-x

Cited by 85 publications

(64 citation statements)

References 40 publications

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“…For the first positive selection, 150 larger colonies were picked up after a 72-h incubation at 37°C and subsequently transferred onto an LB plate containing 100 g of ampicillin per ml and 1 mM IPTG (LB-Amp-IPTG). Halo-forming activities of the cells were analyzed by the Congo red method (11). After growth for 15 h at 37°C, bacterial colonies were overlaid with 10 ml of sterile top agar containing 0.5% CMC and then incubated at 37°C for 6 h to allow hydrolysis of CMC.…”

Section: Vol 66 2000 Screening Of Improved Cellulase Variants 789mentioning

confidence: 99%

“…Whole-cell and free-form CMCase activities were determined according to previous methods (11,19). Enzymatic reactions were performed for 30 min at 37°C with mixing.…”

Section: Vol 66 2000 Screening Of Improved Cellulase Variants 789mentioning

confidence: 99%

“…Recently, a library of surface protease OmpT was displayed, and clones showing improved substrate affinity were isolated by flow cytometry (18). In our study, we have used the ice nucleation protein (Inp)-based bacterial surface display system (10,11) to selectively screen enzyme libraries for improved catalytic activity. Our model enzyme is carboxymethyl cellulase (CMCase).…”

mentioning

confidence: 99%

“…Thus, cells that display CMCase on their surfaces only hydrolyze CMC in agar plates. Cell colonies that hydrolyze CMC can be easily recognized because they are surrounded by a clear halo after staining with Congo red (11). Because Escherichia coli cells displaying CMCases form tiny colonies on M9 minimal medium containing CMC as the sole carbon source, we reasoned that altered growth rates of transformants would be correlated with the activities of displayed CMCase variants.…”

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confidence: 99%

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

Kim

Pan

2000

Appl Environ Microbiol

Self Cite

105

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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.

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Section: Vol 66 2000 Screening Of Improved Cellulase Variants 789mentioning

confidence: 99%

“…Whole-cell and free-form CMCase activities were determined according to previous methods (11,19). Enzymatic reactions were performed for 30 min at 37°C with mixing.…”

Section: Vol 66 2000 Screening Of Improved Cellulase Variants 789mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Kim

Pan

2000

Appl Environ Microbiol

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105

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“…In the full-length INP protein, each major repeat itself consists of 3 repeats of 16 residues (9), which can be depicted as AGYGSTXTAXXXSXLX, where X represents nonconserved residues, and these repeats help in anchoring INP to the outer membrane of the bacterial cell surface. The N-and C-terminal domains have been found to act as better anchoring motifs in comparison to the full-length INP of Pseudomonas syringae (11,20). Full-length INP has been successfully used in the construction of fusion genes for surface expression (11); however, in the present study, full-length INP failed to construct a fusion gene with hPVR.…”

Section: Discussionmentioning

confidence: 65%

Cell Surface Display of Poliovirus Receptor on Escherichia coli, a Novel Method for Concentrating Viral Particles in Water

Abbaszadegan

Alum

Abbaszadegan

et al. 2011

Appl Environ Microbiol

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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.Despite significant improvements in the methodologies for detection of enteric viruses in water samples, there remains a need for more rapid and economical methods. Refined methods that reduce the time and processing involved in concentrating water samples are essential for improved applicability in the detection of viruses. New methods must be compatible with techniques such as cell culturing and molecular analyses. The current USEPA method (26, 27) processes large volumes of water by filtration/adsorption, and elution procedures are used to concentrate and detect enteric viruses in water samples. Over the years, there have been various improvements in detecting enteric viruses by using PCR (1, 2, 8); however, only limited work has been reported on conventional adsorptionelution concentration methods (16). Therefore, there is a need for innovative change in concentration methods, which can be achieved by a paradigm shift in the basic principles.Enteroviruses can cause a variety of illnesses (22), and their presence in source and drinking water (3, 13, 14) and wastewater (24) has been widely documented. Enteroviruses in the environment create a public health risk because they can be transmitted via the fecal-oral route through contaminated water and have been the etiologic agents of many waterborne disease outbreaks (7, 28).The primary objective of this research was to develop a novel strategy that could be used for the detection of viral contamination in water. We engineered an Escherichia coli cell displaying human poliovirus receptor (hPVR) on its cell surface for capturing poliovirus particles in water samples. Cell surface display is a technique that can be used to express heterologous proteins on the outer membranes of bacterial cells, such as E. coli. This technique is widely used in molecular genetics stud...

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Bacterial Display of Antibodies

Blarcom

Harvey²

2009

Therapeutic Monoclonal Antibodies

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Expression of Carboxymethylcellulase on the Surface of Escherichia Coli Using Pseudomonas Syringae Ice Nucleation Protein

Cited by 85 publications

References 40 publications

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

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

Cell Surface Display of Poliovirus Receptor on Escherichia coli, a Novel Method for Concentrating Viral Particles in Water

Bacterial Display of Antibodies

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