Alpha-amylase inhibitors selected from a combinatorial library of a cellulose binding domain scaffold

Lehtiö, Janne; Teeri, Tuula T.; Nygren, Per‐Åke

doi:10.1002/1097-0134(20001115)41:3<316::aid-prot40>3.0.co;2-k

Cited by 43 publications

(33 citation statements)

References 48 publications

(68 reference statements)

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“…Furthermore, single-chain Fv antibody fragments, as well as IgE-and IgA-specific affibody ligands, have been displayed, creating potential whole-cell diagnostic devices (12, 13). In addition, staphylococci with increased metal-binding capacity have been created through display of polyhistidyl peptides (35), and novel metal-binding proteins have been generated through combinatorial protein engineering approaches (19,44 (45). Here, we report the use of a model system employing surface-displayed engineered SpA domains to test the functionality of this S. carnosus display system for the specific enrichment of target cells from a large excess of background cells by flow cytometry.…”

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confidence: 99%

Fluorescence-Activated Cell Sorting of Specific Affibody-Displaying Staphylococci

Wernérus

Samuelson

Ståhl

2003

Appl Environ Microbiol

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Efficient enrichment of staphylococcal cells displaying specific heterologous affinity ligands on their cell surfaces was demonstrated by using fluorescence-activated cell sorting. Using bacterial surface display of peptide or protein libraries for the purpose of combinatorial protein engineering has previously been investigated by using gram-negative bacteria. Here, the potential for using a gram-positive bacterium was evaluated by employing the well-established surface expression system for Staphylococcus carnosus. Staphylococcus aureus protein A domains with binding specificity to immunoglobulin G or engineered specificity for the G protein of human respiratory syncytial virus were expressed as surface display on S. carnosus cells. The surface accessibility and retained binding specificity of expressed proteins were demonstrated in whole-cell enzyme and flow cytometry assays. Also, affibody-expressing target cells could be sorted essentially quantitatively from a moderate excess of background cells in a single step by using a high-stringency sorting mode. Furthermore, in a simulated library selection experiment, a more-than-25,000-fold enrichment of target cells could be achieved through only two rounds of cell sorting and regrowth. The results obtained indicate that staphylococcal surface display of affibody libraries combined with fluoresence-activated cell sorting might indeed constitute an attractive alternative to existing technology platforms for affinity-based selections.Recent advances within the field of combinatorial protein engineering have led to the development of several complementary technologies for the selection of novel protein variants from large libraries. So far, phage display has been the preferred format for directed evolution efforts (39), but more recently, other techniques, such as ribosomal display (29), covalent display, and different formats of cell display, have become attractive alternatives (46). Cell surface display combined with fluorescence-activated cell sorting (FACS) constitutes a powerful strategy for isolation of novel ligands with improved affinity, stability, or enzymatic activity (4,28,37). The high throughput and quantitative multiparameter population analysis of modern flow cytometers makes FACS ideal for protein engineering applications (7,46). However, FACS sorting would only be applicable with cell display systems, since the phage particles are too small to be sorted with the present state-of-the-art flow cytometers (6, 43).Traditionally, engineering of antibody fragments has been the dominating strategy for generating novel proteins with specific ligand-binding properties (17). More recently, other protein scaffolds have also been investigated as sources for novel affinity ligands (26,38). One such novel class of affinity proteins, called affibody ligands, i.e., engineered Staphylococcus aureus protein A (SpA) domains, has recently been described (23). Combinatorial libraries were created through simultaneous randomization of 13 amino acid residues. Through the...

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confidence: 99%

Fluorescence-Activated Cell Sorting of Specific Affibody-Displaying Staphylococci

Wernérus

Samuelson

Ståhl

2003

Appl Environ Microbiol

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“…E. coli cells, approximately 10 9 cells of bacterial strain RRI⌬M15 (40), containing the constructed CBD library (23), were added to 100 ml of TSB medium supplemented with 100 mg of ampicillin per liter and 2% glucose, incubated at 37°C to an A 600 nm of Ϸ0.5, and then infected with helper phage M13K07 (5 ϫ 10 10 particles) (New England Biolabs) at 37°C for 30 min. Superinfected cells were harvested and used to inoculate TSB medium supplemented with ampicillin (100 mg/liter), kanamycin (25 mg/liter), and isopropyl-␤-D-thiogalactoside (IPTG) (100 M).…”

Section: Methodsmentioning

confidence: 99%

“…Recently, a fungal cellulose-binding domain (CBD) derived from the cellobiohydrolase Cel7A of Trichoderma reesei was subjected to a combinatorial protein engineering approach (23). A combinatorial library comprising 46 million variants of the 36-amino-acid CBD domain was constructed through the randomization of 11 amino acid positions, including the residues involved in cellulose binding.…”

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confidence: 99%

“…A combinatorial library comprising 46 million variants of the 36-amino-acid CBD domain was constructed through the randomization of 11 amino acid positions, including the residues involved in cellulose binding. Using phage display technology, CBD variants that showed specific binding of and ability to inhibit the target enzyme porcine ␣-amylase (PPA) could be selected (23). Furthermore, a related CBD, derived from T. reesei cellulase Cel6A, was recently expressed in its nonengineered form on the surface of S. carnosus (24).…”

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“…The proven capacity of CBD to be engineered (23) and displayed on the surface of bacteria (24) inspired us to investigate the possibility of using the CBD scaffold for metal capture as well. In this study we have evaluated a strategy to generate bacteria with an increased affinity for nickel ions by combining phage display-based combinatorial protein engineering with subsequent surface expression on S. carnosus cells.…”

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Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

Wernérus

Lehtiö

Teeri

et al. 2001

Appl Environ Microbiol

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Ni2؉ -binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reesei cellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni 2؉ -binding CBDs were selected by phage display technology. These new variants were subsequently genetically introduced into chimeric surface proteins for surface display on Staphylococcus carnosus cells. The expressed chimeric proteins were shown to be properly targeted to the cell wall of S. carnosus cells, since full-length proteins could be extracted and affinity purified. Surface accessibility for the chimeric proteins was demonstrated, and furthermore, the engineered CBDs, now devoid of cellulose-binding capacity, were shown to be functional with regard to metal binding, since the recombinant staphylococci had gained Ni 2؉ -binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.Bacterial surface display of heterologous proteins has in recent years become an increasingly active research area with a wide range of applications in immunology, vaccinology, and biotechnology (11, 49). An interesting application area is the display of metal-binding proteins on bacteria to create wholecell tools for improved sequestration of toxic metals in wastewater (3), a field in which naturally occurring bacteria have been evaluated previously (10, 31). The heterologous expression of peptides and proteins with inherent metal-binding capacity have been employed to create bacteria with improved metalloadsorption properties (39), and surface display has been used as an attractive strategy in this context (22,38,47,51).Short metal-binding peptides, such as hexahistidyl peptides, have been introduced into bacterial surface proteins in order to create bacteria with improved metal-binding capacity (21,46). Using this strategy, both Escherichia coli (46) and Staphylococcus carnosus (43) strains with increased ability to bind Ni 2ϩ and Cd 2ϩ ions have been generated. The introduction of combinatorial protein engineering has also made it possible to select peptides, from large peptide libraries with increased selectivity for certain metals (4,30,37,44), and such peptides might become interesting for surface display applications (44).Gram-positive surface display systems have been suggested to exhibit some advantages compared to gram-negative bacteria (28, 49): (i) the translocation involves only a single membrane, and (ii) gram-positive bacteria have been shown to be more rigid and thus less sensitive to shear forces (19, 36) due to the thick cell wall surrounding the cells, and thus potentially more suitable for field applications such as bioadsorption. For metal adsorption applications, gram-positive bacteria have the additional advantage of having inherent metal-binding capacity due to the thick pe...

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Non‐Antibody Scaffolds

Fiedler¹,

Skerra²

2007

Handbook of Therapeutic Antibodies

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Alpha-amylase inhibitors selected from a combinatorial library of a cellulose binding domain scaffold

Cited by 43 publications

References 48 publications

Fluorescence-Activated Cell Sorting of Specific Affibody-Displaying Staphylococci

Fluorescence-Activated Cell Sorting of Specific Affibody-Displaying Staphylococci

Generation of Metal-Binding Staphylococci through Surface Display of Combinatorially Engineered Cellulose-Binding Domains

Non‐Antibody Scaffolds

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