Cell‐Surface display of heterologous proteins: From high‐throughput screening to environmental applications

Chen, Wilfred; Georgiou, George

doi:10.1002/bit.10407

Cited by 106 publications

(66 citation statements)

References 87 publications

(71 reference statements)

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“…Display of enzymes on the surface of microorganisms such as E. coli or yeast can be accomplished in a variety of ways (32,33). In this manner, the enzyme can react with exogenous synthetic substrates, circumventing the substrate transport limitations associated with methods that use intracellularly expressed enzymes.…”

Section: Discussionmentioning

confidence: 99%

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Varadarajan

Gam

Olsen

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

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138

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The exquisite selectivity and catalytic activity of enzymes have been shaped by the effects of positive and negative selection pressure during the course of evolution. In contrast, enzyme variants engineered by using in vitro screening techniques to accept novel substrates typically display a higher degree of catalytic promiscuity and lower total turnover in comparison with their natural counterparts. Using bacterial display and multiparameter flow cytometry, we have developed a novel methodology for emulating positive and negative selective pressure in vitro for the isolation of enzyme variants with reactivity for desired novel substrates, while simultaneously excluding those with reactivity toward undesired substrates. Screening of a large library of random mutants of the Escherichia coli endopeptidase OmpT led to the isolation of an enzyme variant, 1.3.19, that cleaved an Ala-Arg peptide bond instead of the Arg-Arg bond preferred by the WT enzyme. Variant 1.3.19 exhibited greater than three million-fold selectivity (-Ala-Arg-͞-Arg-Arg-) and a catalytic efficiency for AlaArg cleavage that is the same as that displayed by the parent for the preferred substrate, Arg-Arg. A single amino acid Ser223Arg substitution was shown to recapitulate completely the unique catalytic properties of the 1.3.19 variant. These results can be explained by proposing that this mutation acts to ''swap'' the P 1 Arg side chain normally found in WT substrate peptides with the 223Arg side chain in the S 1 subsite of OmpT.engineering ͉ flow cytometry T he reprogramming of enzyme catalytic activity and selectivity is a central issue in protein biochemistry and biotechnology. Numerous structure-guided and directed evolution strategies have been used in search of enzyme variants that exhibit high catalytic rates with poor or inactive substrates of the parental enzyme (1-19). As impressive as these successes have been, the engineering of enzymes that exhibit turnover rates and selectivities with new substrates comparable to their natural counterparts has proven quite a challenge, especially when considering those enzymes for which a genetic selection strategy is not possible.In particular, enzymes engineered through laboratory evolution involving in vitro catalytic assays have often been found lacking, either with respect to turnover rates or selectivity, relative to catalyst-substrate pairs isolated from natural sources. As a typical example, an extensive directed evolution program led to the isolation of Escherichia coli ␤-glucuronidase variants with significant ␤-galactosidase (10) or xylanosidase (11) activities, but nonetheless even the best clones exhibited k cat ͞K m values Ͼ1,000 times lower than those of naturally occurring enzymes such as the E. coli ␤-galactosidase or the Thermoanaerobacterium saccharolyticum ␤-xylosidase.This trend appears to be general. In a recent comprehensive study, Aaron et al. (12) demonstrated that the evolution of higher activity toward poor substrates did not impair the parental catalytic activity, and,...

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Section: Discussionmentioning

confidence: 99%

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Varadarajan

Gam

Olsen

et al. 2005

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

138

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“…They have proved quite useful in a broad range of fields, including protein engineering, screening for antibody fragments, whole cell catalysts, adsorbents for bioremediation, etc. (112,113). A major limitation, however, is there are only 20 naturallyoccurring amino acids.…”

Section: Tagging E Colimentioning

confidence: 99%

Click Chemistry, A Powerful Tool for Pharmaceutical Sciences

2008

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Abstract. Click chemistry refers to a group of reactions that are fast, simple to use, easy to purify, versatile, regiospecific, and give high product yields. While there are a number of reactions that fulfill the criteria, the Huisgen 1,3-dipolar cycloaddition of azides and terminal alkynes has emerged as the frontrunner. It has found applications in a wide variety of research areas, including materials sciences, polymer chemistry, and pharmaceutical sciences. In this manuscript, important aspects of the Huisgen cycloaddition will be reviewed, along with some of its many pharmaceutical applications. Bioconjugation, nanoparticle surface modification, and pharmaceutical-related polymer chemistry will all be covered. Limitations of the reaction will also be discussed.

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“…Fusion proteins are known to produce incorrect tertiary structures that affect the topicity of expressed proteins and may cause steric hindrance, thus rendering the proteins nonfunctional in the docking process (6). These issues were addressed by investigating the docking capability of poliovirus particles with the recombinant E. coli.…”

Section: Vol 77 2011 Engineered E Coli For Poliovirus Recovery 5145mentioning

confidence: 99%

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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Cell‐Surface display of heterologous proteins: From high‐throughput screening to environmental applications

Cited by 106 publications

References 87 publications

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Engineering of protease variants exhibiting high catalytic activity and exquisite substrate selectivity

Click Chemistry, A Powerful Tool for Pharmaceutical Sciences

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

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