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

ABSTRACT Ni2+-binding staphylococci were generated through surface display of combinatorially engineered variants of a fungal cellulose-binding domain (CBD) from Trichoderma reeseicellulase Cel7A. Novel CBD variants were generated by combinatorial protein engineering through the randomization of 11 amino acid positions, and eight potentially Ni2+-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 Ni2+-binding capacity. Potential environmental applications for such tailor-made metal-binding bacteria as bioadsorbents in biofilters or biosensors are discussed.

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Staphylococcal Surface Display of Metal-Binding Polyhistidyl Peptides

Applied and Environmental Microbiology ◽

10.1128/aem.66.3.1243-1248.2000 ◽

2000 ◽

Vol 66 (3) ◽

pp. 1243-1248 ◽

Cited By ~ 78

Author(s):

Patrik Samuelson ◽

Henrik Wernérus ◽

Malin Svedberg ◽

Stefan Ståhl

Keyword(s):

Metal Binding ◽

Binding Capacity ◽

Surface Display ◽

Surface Proteins ◽

Chimeric Proteins ◽

Divalent Metal Ions ◽

Gram Positive Bacteria ◽

Metal Binding Peptides ◽

Binding Peptides ◽

First Time

ABSTRACT Recombinant Staphylococcus xylosus andStaphylococcus carnosus strains were generated with surface-exposed chimeric proteins containing polyhistidyl peptides designed for binding to divalent metal ions. Surface accessibility of the chimeric surface proteins was demonstrated and the chimeric surface proteins were found to be functional in terms of metal binding, since the recombinant staphylococcal cells were shown to have gained Ni2+- and Cd2+-binding capacity, suggesting that such bacteria could find use in bioremediation of heavy metals. This is, to our knowledge, the first time that recombinant, surface-exposed metal-binding peptides have been expressed on gram-positive bacteria. Potential environmental or biosensor applications for such recombinant staphylococci as biosorbents are discussed.

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Characterization of hybrid proteins consisting of the catalytic domains of Clostridium and Ruminococcus endoglucanases, fused to Pseudomonas non-catalytic cellulose-binding domains

Biochemical Journal ◽

10.1042/bj2790787 ◽

1991 ◽

Vol 279 (3) ◽

pp. 787-792 ◽

Cited By ~ 22

Author(s):

D M Poole ◽

A J Durrant ◽

G P Hazlewood ◽

H J Gilbert

Keyword(s):

Catalytic Domain ◽

Binding Capacity ◽

Binding Domains ◽

Hybrid Proteins ◽

C Terminus ◽

N Terminus ◽

Fusion Enzymes ◽

Cellulose Binding

The N-terminal 160 or 267 residues of xylanase A from Pseudomonas fluorescens subsp. cellulosa, containing a non-catalytic cellulose-binding domain (CBD), were fused to the N-terminus of the catalytic domain of endoglucanase E (EGE') from Clostridium thermocellum. A further hybrid enzyme was constructed consisting of the 347 N-terminal residues of xylanase C (XYLC) from P. fluorescens subsp. cellulosa, which also constitutes a CBD, fused to the N-terminus of endoglucanase A (EGA) from Ruminococcus albus. The three hybrid enzymes bound to insoluble cellulose, and could be eluted such that cellulose-binding capacity and catalytic activity were retained. The catalytic properties of the fusion enzymes were similar to EGE' and EGA respectively. Residues 37-347 and 34-347 of XYLC were fused to the C-terminus of EGE' and the 10 amino acids encoded by the multiple cloning sequence of pMTL22p respectively. The two hybrid proteins did not bind cellulose, although residues 39-139 of XYLC were shown previously to constitute a functional CBD. The putative role of the P. fluorescens subsp. cellulosa CBD in cellulase action is discussed.

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Staphylococcal Surface Display of Immunoglobulin A (IgA)- and IgE-Specific In Vitro-Selected Binding Proteins (Affibodies) Based on Staphylococcus aureus Protein A

Applied and Environmental Microbiology ◽

10.1128/aem.65.9.4134-4140.1999 ◽

1999 ◽

Vol 65 (9) ◽

pp. 4134-4140 ◽

Cited By ~ 35

Author(s):

Elin Gunneriusson ◽

Patrik Samuelson ◽

Jenny Ringdahl ◽

Hans Grönlund ◽

Per-Åke Nygren ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Cell Surface ◽

Binding Capacity ◽

Expression System ◽

Surface Display ◽

Protein A ◽

Surface Proteins ◽

Phage Library ◽

Protein Chemistry ◽

Ige Binding

ABSTRACT An expression system designed for cell surface display of hybrid proteins on Staphylococcus carnosus has been evaluated for the display of Staphylococcus aureus protein A (SpA) domains, normally binding to immunoglobulin G (IgG) Fc but here engineered by combinatorial protein chemistry to yield SpA domains, denoted affibodies, with new binding specificities. Such affibodies, with human IgA or IgE binding activity, have previously been selected from a phage library, based on an SpA domain. In this study, these affibodies have been genetically introduced in monomeric or dimeric forms into chimeric proteins expressed on the surface of S. carnosus by using translocation signals from aStaphylococcus hyicus lipase construct together with surface-anchoring regions of SpA. The recombinant surface proteins, containing the IgA- or IgE-specific affibodies, were demonstrated to be expressed as full-length proteins, localized and properly exposed at the cell surface of S. carnosus. Furthermore, these chimeric receptors were found to be functional, since recombinantS. carnosus cells were shown to have gained IgA and IgE binding capacity, respectively. In addition, a positive effect in terms of IgA and IgE reactivity was observed when dimeric versions of the affibodies were present. Potential applications for recombinant bacteria with redirected binding specificity in their surface proteins are discussed.

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Spatial separation of protein domains is not necessary for catalytic activity or substrate binding in a xylanase

Biochemical Journal ◽

10.1042/bj2690261 ◽

1990 ◽

Vol 269 (1) ◽

pp. 261-264 ◽

Cited By ~ 55

Author(s):

L M A Ferreira ◽

A J Durrant ◽

J Hall ◽

G P Hazlewood ◽

H J Gilbert

Keyword(s):

Catalytic Activity ◽

Specific Activity ◽

Binding Capacity ◽

Spatial Separation ◽

Crystalline Cellulose ◽

Conserved Sequence ◽

Binding Domains ◽

Catalytically Active ◽

Cellulose Binding ◽

Derivatives Of

Xylanase A (XYLA) from Pseudomonas fluorescens subspecies cellulosa shows sequence conservation with two endoglucanases from the same organism. The conserved sequence in XYLA, consisting of the N-terminal 234 residues, is not essential for catalytic activity. Full-length XYLA and a fusion enzyme, consisting of the N-terminal 100 residues of XYLA linked to mature alkaline phosphatase, bound tightly to crystalline cellulose (Avicel), but not to xylan. The capacity of truncated derivatives of the xylanase to bind polysaccharides was investigated. XYLA lacking the first 13 N-terminal amino acids did not bind to cellulose. However, a catalytically active XYLA derivative (XYLA′), in which residues 100-234 were deleted, bound tightly to Avicel. Substrate specificity, cellulose-binding capacity, specific activity and Km for xylan hydrolysis were evaluated for each of the xylanases. No differences in any of these parameters were detected for the two enzymes. It is concluded that XYLA contains a cellulose-binding domain consisting of the N-terminal 100 residues which is distinct from the active site. Spatial separation of the catalytic and cellulose-binding domains is not essential for the enzyme to function normally.

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The first report on the sortase-mediated display of bioactive protein A from Staphylococcus aureus (SpA) on the surface of the vegetative form of Bacillus subtilis

Microbial Cell Factories ◽

10.1186/s12934-021-01701-4 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Samira Ghaedmohammadi ◽

Gholamreza Ahmadian

Keyword(s):

Staphylococcus Aureus ◽

Cell Wall ◽

Bacillus Subtilis ◽

Binding Capacity ◽

Surface Display ◽

Protein A ◽

Production Costs ◽

Rabbit Serum ◽

Experimental Conditions ◽

Binding Domains

AbstractProtein A (SpA) is one of the most important Staphylococcus aureus cell wall proteins. It includes five immunoglobulin (Ig)-binding domains which can bind to immune complexes through the Fc region of immunoglobulins. The binding of SpA to the polymeric supports can be used to prepare affinity chromatography resins, which are useful for immunoprecipitation (IP) of antibodies. Protein A is also used to purify many anti-cancer antibodies. In this study, SpA was displayed on the surface of Bacillus subtilis cells using a sortase-mediated system to display the target protein to the B. subtilis cell wall. A series of plasmids consisting of cassettes for cell wall-directed protein A as well as negative controls were constructed and transformed into B. subtilis WASD (wprA sigD) cells. SDS-PAGE, western blot, flow cytometry, functional IgG purification assay, and a modified ELISA assay were used to confirm the surface display of SpA and evaluate its function. Semi-quantitative ELISA results showed that the binding capacity of lyophilized Bs-SpA is 100 μg IgG from rabbit serum per 1 mg of cells under optimal experimental conditions. Low production costs, optimal performance, and the use of a harmless strain compared to a similar commercial product predict the possible use of SpA immobilization technology in the future.

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Enhanced Bioaccumulation of Heavy Metal Ions by Bacterial Cells Due to Surface Display of Short Metal Binding Peptides

Applied and Environmental Microbiology ◽

10.1128/aem.65.3.1092-1098.1999 ◽

1999 ◽

Vol 65 (3) ◽

pp. 1092-1098 ◽

Cited By ~ 125

Author(s):

Pavel Kotrba ◽

Lucie Dolečková ◽

Víctor de Lorenzo ◽

Tomas Ruml

Keyword(s):

Metal Binding ◽

Metal Ion ◽

Rational Design ◽

Binding Capacity ◽

Surface Display ◽

Genetically Engineered ◽

E Coli ◽

Metal Binding Peptides ◽

Binding Peptides ◽

Cell Envelopes

ABSTRACT Metal binding peptides of sequences Gly-His-His-Pro-His-Gly (named HP) and Gly-Cys-Gly-Cys-Pro-Cys-Gly-Cys-Gly (named CP) were genetically engineered into LamB protein and expressed in Escherichia coli. The Cd2+-to-HP and Cd2+-to-CP stoichiometries of peptides were 1:1 and 3:1, respectively. Hybrid LamB proteins were found to be properly folded in the outer membrane ofE. coli. Isolated cell envelopes of E. colibearing newly added metal binding peptides showed an up to 1.8-fold increase in Cd2+ binding capacity. The bioaccumulation of Cd2+, Cu2+, and Zn2+ by E. coli was evaluated. Surface display of CP multiplied the ability of E. coli to bind Cd2+ from growth medium fourfold. Display of HP peptide did not contribute to an increase in the accumulation of Cu2+ and Zn2+. However, Cu2+ ceased contribution of HP for Cd2+accumulation, probably due to the strong binding of Cu2+ to HP. Thus, considering the cooperation of cell structures with inserted peptides, the relative affinities of metal binding peptide and, for example, the cell wall to metal ion should be taken into account in the rational design of peptide sequences possessing specificity for a particular metal.

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Fusion of family VI cellulose binding domains to Bacillus halodurans xylanase increases its catalytic activity and substrate-binding capacity to insoluble xylan

Journal of Molecular Catalysis B Enzymatic ◽

10.1016/s1381-1177(02)00226-6 ◽

2003 ◽

Vol 21 (4-6) ◽

pp. 221-230 ◽

Cited By ~ 36

Author(s):

S.L. Mangala ◽

F.S. Kittur ◽

M. Nishimoto ◽

K. Sakka ◽

K. Ohmiya ◽

...

Keyword(s):

Catalytic Activity ◽

Binding Capacity ◽

Substrate Binding ◽

Bacillus Halodurans ◽

Binding Domains ◽

Cellulose Binding

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Chimeric Proteins Combining Phosphatase and Cellulose-Binding Activities: Proof-of-Concept and Application in the Hydrolysis of Paraoxon

Protein and Peptide Letters ◽

10.2174/092986652105140218114537 ◽

2014 ◽

Vol 21 (5) ◽

pp. 468-475 ◽

Cited By ~ 1

Author(s):

Larissa Goncalves ◽

Hernan Chaimovich ◽

Iolanda Cuccovia ◽

Sandro Marana

Keyword(s):

Chimeric Proteins ◽

Proof Of Concept ◽

Cellulose Binding ◽

Hydrolysis Of

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Development and Application of Computational Methods in Phage Display Technology

Current Medicinal Chemistry ◽

10.2174/0929867325666180629123117 ◽

2020 ◽

Vol 26 (42) ◽

pp. 7672-7693 ◽

Cited By ~ 3

Author(s):

Bifang He ◽

Anthony Mackitz Dzisoo ◽

Ratmir Derda ◽

Jian Huang

Keyword(s):

Phage Display ◽

Computational Methods ◽

Peptide Ligands ◽

Next Generation ◽

Phage Display Technology ◽

Next Generation Sequencing Technology ◽

Screening Experiments ◽

Display Technology ◽

Comprehensive Search ◽

Generation Sequencing

Background: Phage display is a powerful and versatile technology for the identification of peptide ligands binding to multiple targets, which has been successfully employed in various fields, such as diagnostics and therapeutics, drug-delivery and material science. The integration of next generation sequencing technology with phage display makes this methodology more productive. With the widespread use of this technique and the fast accumulation of phage display data, databases for these data and computational methods have become an indispensable part in this community. This review aims to summarize and discuss recent progress in the development and application of computational methods in the field of phage display. Methods: We undertook a comprehensive search of bioinformatics resources and computational methods for phage display data via Google Scholar and PubMed. The methods and tools were further divided into different categories according to their uses. Results: We described seven special or relevant databases for phage display data, which provided an evidence-based source for phage display researchers to clean their biopanning results. These databases can identify and report possible target-unrelated peptides (TUPs), thereby excluding false-positive data from peptides obtained from phage display screening experiments. More than 20 computational methods for analyzing biopanning data were also reviewed. These methods were classified into computational methods for reporting TUPs, for predicting epitopes and for analyzing next generation phage display data. Conclusion: The current bioinformatics archives, methods and tools reviewed here have benefitted the biopanning community. To develop better or new computational tools, some promising directions are also discussed.

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