Display of heterologous proteins on the surface of microorganisms: From the screening of combinatorial libraries to live recombinant vaccines

Georgiou, George; Stathopoulos, Christos; Daugherty, Patrick S.; Nayak, Amiya R.; Iverson, Brent L.; Curtiss, Roy

doi:10.1038/nbt0197-29

Cited by 483 publications

(322 citation statements)

References 77 publications

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“…This approach relies on fluorescent substrates of different colors that label the surface of E. coli cells upon cleavage by a surface-anchored enzyme (20). Enzymes can be displayed on the surface of Gram-negative bacteria by established techniques (21,22), and thus, access to the fluorescent substrate is assured. The net result is that the cell fluorescence profile accurately reflects the catalytic activity and selectivity of the surface-displayed enzyme.…”

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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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137

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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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mentioning

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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137

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“…For screening, we used noninfectable F À E. coli HB101 cells carrying single-chain antibody fragments (scFv) scFv(225) of cetuximab (Wels et al, 1995) or scFv(72000) of matuzumab as Lpp-OmpA' fusions (Georgiou et al, 1997) on the surface as schematically shown in Figures 1a and b. After removal of unbound phage particles, bound phage were amplified by adding infectable F þ E. coli ER2738.…”

Section: Resultsmentioning

confidence: 99%

Peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-EGFR immune response

et al. 2010

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“…Surface display of foreign epitopes on bacteria can be useful in many applications, such as recombinant vaccines, reagents for diagnostics, whole-cell biocatalysts and bioadsorbants (Georgiou et al, 1997). Over the last decade, a range of immune-relevant sectors of foreign proteins have been successfully displayed on the bacterial cell surface by a large variety of fimbriae and flagella.…”

Section: Discussionmentioning

confidence: 99%

DAF- and collagen-binding properties of chimeric Dr fimbriae

et al. 2007

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Display of heterologous proteins on the surface of micro-organisms has become an increasingly used strategy for a range of applications in microbiology, biotechnology and vaccinology. In this study, the potential of the major structural protein DraE of Escherichia coli Dr fimbriae as a display system for heterologous sequences was tested. One copy of a heterologous sequence mimicking a small P k epitope of simian virus 5 was inserted into the draE gene, replacing the N-terminal region of the surface-exposed domain 2 as previously done with the glycoprotein D of herpes simplex virus type 1. The exposure of chimeric proteins on the bacterial surface was detected by immunofluorescence microscopy. Insertion of the heterogenic peptides had no detectable effect on the Ig-barrel structure of the DraE fimbrial subunits, as confirmed by FTIR spectroscopy. Additionally, the affinity of the chimeric fimbriae for DAF and type IV collagen was similar to that of the wild-type Dr fimbriae.

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Display of heterologous proteins on the surface of microorganisms: From the screening of combinatorial libraries to live recombinant vaccines

Cited by 483 publications

References 77 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

Peptide mimotopes recognized by antibodies cetuximab and matuzumab induce a functionally equivalent anti-EGFR immune response

DAF- and collagen-binding properties of chimeric Dr fimbriae

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