S-Layer-Mediated Display of the Immunoglobulin G-Binding Domain of Streptococcal Protein G on the Surface of<i>Caulobacter crescentus</i>: Development of an Immunoactive Reagent

Nomellini, John F.; Duncan, Graeme; Dorocicz, Irene; Smit, John

doi:10.1128/aem.02900-06

Cited by 54 publications

(60 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…All PCR products were verified by DNA sequencing. The plasmid constructs were introduced into T. pseudonana cells by microparticle bombardment using an established method 25,40 . Transformant clones were selected on agar plates containing the antibiotic nourseothricin.…”

Section: Methodsmentioning

confidence: 99%

Targeted drug delivery using genetically engineered diatom biosilica

et al. 2015

View full text Add to dashboard Cite

The ability to selectively kill cancerous cell populations while leaving healthy cells unaffected is a key goal in anticancer therapeutics. The use of nanoporous silica-based materials as drug-delivery vehicles has recently proven successful, yet production of these materials requires costly and toxic chemicals. Here we use diatom microalgae-derived nanoporous biosilica to deliver chemotherapeutic drugs to cancer cells. The diatom Thalassiosira pseudonana is genetically engineered to display an IgG-binding domain of protein G on the biosilica surface, enabling attachment of cell-targeting antibodies. Neuroblastoma and B-lymphoma cells are selectively targeted and killed by biosilica displaying specific antibodies sorbed with drug-loaded nanoparticles. Treatment with the same biosilica leads to tumour growth regression in a subcutaneous mouse xenograft model of neuroblastoma. These data indicate that genetically engineered biosilica frustules may be used as versatile 'backpacks' for the targeted delivery of poorly water-soluble anticancer drugs to tumour sites.

show abstract

Section: Methodsmentioning

confidence: 99%

Targeted drug delivery using genetically engineered diatom biosilica

et al. 2015

View full text Add to dashboard Cite

show abstract

“…The annealed JN CYS N-1 and JN CYS N-2 oligonucleotides were then ligated into the BglII site. The modified versions of rsaA were transferred to p4A as EcoRI/ HindIII segments (36). The resulting plasmids were introduced into JS1023 by electroporation.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

et al. 2010

Self Cite

View full text Add to dashboard Cite

The surface layers (S layers) of those bacteria and archaea that elaborate these crystalline structures have been studied for 40 years. However, most structural analysis has been based on electron microscopy of negatively stained S-layer fragments separated from cells, which can introduce staining artifacts and allow rearrangement of structures prone to self-assemble. We present a quantitative analysis of the structure and organization of the S layer on intact growing cells of the Gram-negative bacterium Caulobacter crescentus using cryo-electron tomography (CET) and statistical image processing. Instead of the expected long-range order, we observed different regions with hexagonally organized subunits exhibiting short-range order and a broad distribution of periodicities. Also, areas of stacked double layers were found, and these increased in extent when the S-layer protein (RsaA) expression level was elevated by addition of multiple rsaA copies. Finally, we combined high-resolution amino acid residue-specific Nanogold labeling and subtomogram averaging of CET volumes to improve our understanding of the correlation between the linear protein sequence and the structure at the 2-nm level of resolution that is presently available. The results support the view that the U-shaped RsaA monomer predicted from negative-stain tomography proceeds from the N terminus at one vertex, corresponding to the axis of 3-fold symmetry, to the C terminus at the opposite vertex, which forms the prominent 6-fold symmetry axis. Such information will help future efforts to analyze subunit interactions and guide selection of internal sites for display of heterologous protein segments.Surface layers (S layers) are the outermost cell wall component in many archaea and bacteria (6, 44). Most S layers are composed of a single protein or glycoprotein species that selforganizes into two-dimensional (2D) lattices of various sizes, usually with square or hexagonal symmetry (7,14,43). This geometrical arrangement is almost the only commonality among species, since sequence homology between S-layer proteins is low and functionality differs in many cases. In many archaea, the S layer is the only cell wall component, so it may have a role in shape determination. However, in bacteria such as Caulobacter crescentus, the role is more likely related to protection against a variety of predatorial assaults (8).One interest in understanding S layers comes from their potential applications in nanotechnology (46) and therapeutic applications, such as anti-HIV microbicide development (37) and cancer therapy (9). The concept is to display heterologous proteins from within the S-layer structure in order to create dense arrays of foreign insertions. Resolving the S-layer organization and structure at high resolution in cells as close to a native state as possible is crucial to understand or predict where proteins are displayed in the array, particularly when more than one foreign peptide is being displayed simultaneously.A significant limitation has been the dif...

show abstract

“…The S-layer protein of Caulobacter crescentus, RsaA, was used to express protein G on the surface of C. crescentus cells [53]. This will provide a low-cost method for antibody binding, with applications in the development of immunoassays.…”

Section: S-layer Proteins Have Been Detected Across All Phylogentic Gmentioning

confidence: 99%

Versatile microbial surface-display for environmental remediation and biofuels production

Wu¹,

Mulchandani²,

Chen³

2008

Trends in Microbiology

View full text Add to dashboard Cite

Surface display is a powerful technique that utilizes natural microbial functional components to express proteins or peptides on the cell exterior. Since the reporting of the first surface-display system in the mid-1980s, a variety of new systems have been reported for yeast, Gram-positive and Gram-negative bacteria. Non-conventional display methods are emerging, eliminating the generation of genetically modified microorganisms. Cells with surface display are used as biocatalysts, biosorbents and biostimulants. Microbial cell-surface display has proven to be extremely important for numerous applications ranging from combinatorial library screening and protein engineering to bioremediation and biofuels production.3

show abstract

S-Layer-Mediated Display of the Immunoglobulin G-Binding Domain of Streptococcal Protein G on the Surface ofCaulobacter crescentus: Development of an Immunoactive Reagent

Cited by 54 publications

References 36 publications

Targeted drug delivery using genetically engineered diatom biosilica

Targeted drug delivery using genetically engineered diatom biosilica

Analysis of the Intact Surface Layer of Caulobacter crescentus by Cryo-Electron Tomography

Versatile microbial surface-display for environmental remediation and biofuels production

Contact Info

Product

Resources

About