Early sensitive diagnosis of cancer is critical for enhancing treatment success. We previously bioengineered multifunctional core–shell structures composed of a poly-3-hydroxybutyrate (PHB) core ...densely coated with protein functions for uses in bioseparation and immunodiagnostic applications. Here, we report bioengineering of Escherichia coli to self-assemble PHB inclusions that codisplay a ferritin-derived iron-binding peptide and the protein A-derived antibody-binding Z domain. The iron-binding peptide mediated surface coating with a ferrofluid imparting superparamagnetic properties, while the Z domain remained accessible for binding of cancer biomarker-specific antibodies. We demonstrated that these nanobeads can specifically bind biomarkers in complex mixtures, enabling efficient magnetic separation toward enhanced electrochemical detection of cancer biomarkers such as methylated DNA and exosomes from cancer cells. Our study revealed that superparamagnetic core–shell structures can be derived from biological self-assembly systems for uses in sensitive and specific electrochemical detection of cancer biomarkers, laying the foundation for engineering advanced nanomaterials for diverse diagnostic approaches.
The exopolysaccharide alginate, produced by the opportunistic human pathogen
, confers a survival advantage to the bacterium by contributing to the formation of characteristic biofilms during ...infection. Membrane-anchored proteins Alg8 (catalytic subunit) and Alg44 (copolymerase) constitute the alginate polymerase that is being activated by the second messenger molecule bis-(3', 5')-cyclic dimeric GMP (c-di-GMP), but the mechanism of activation remains elusive. To shed light on the c-di-GMP-mediated activation of alginate polymerization
, an
structural model of Alg8 fused to the c-di-GMP binding PilZ domain informed by the structure of cellulose synthase, BcsA, was developed. This structural model was probed by site-specific mutagenesis and different cellular levels of c-di-GMP. Results suggested that c-di-GMP-mediated activation of alginate polymerization involves amino acids residing at two loops, including H323 (loop A) and T457 and E460 (loop B), surrounding the catalytic site in the predicted model. The activities of the respective Alg8 variants suggested that c-di-GMP-mediated control of substrate access to the catalytic site of Alg8 is dissimilar to the known activation mechanism of BcsA. Alg8 variants responded differently to various c-di-GMP levels, while MucR imparted c-di-GMP for activation of alginate polymerase. Furthermore, we showed that Alg44 copolymerase constituted a stable dimer, with its periplasmic domains required for protein localization and alginate polymerization and modification. Superfolder green fluorescent protein (GFP) fusions of Alg8 and Alg44 showed a nonuniform, punctate, and patchy arrangement of both proteins surrounding the cell. Overall, this study provides insights into the c-di-GMP-mediated activation of alginate polymerization while assigning functional roles to Alg8 and Alg44, including their subcellular localization and distribution.
The exopolysaccharide alginate is an important biofilm component of the opportunistic human pathogen
and the principal cause of the mucoid phenotype that is the hallmark of chronic infections of cystic fibrosis patients. The production of alginate is mediated by interacting membrane proteins Alg8 and Alg44, while their activity is posttranslationally regulated by the second messenger c-di-GMP, a well-known regulator of the synthesis of a range of other exopolysaccharides in bacteria. This study provides new insights into the unknown activation mechanism of alginate polymerization by c-di-GMP. Experimental evidence that the activation of alginate polymerization requires the engagement of specific amino acid residues residing at the catalytic domain of Alg8 glycosyltransferase was obtained, and these residues are proposed to exert an allosteric effect on the PilZ
domain upon c-di-GMP binding. This mechanism is dissimilar to the proposed mechanism of the autoinhibition of cellulose polymerization imposed by salt bridge formation between amino acid residues and released upon c-di-GMP binding, leading to activation of polymerization. On the other hand, conserved amino acid residues in the periplasmic domain of Alg44 were found to be involved in alginate polymerization as well as modification events, i.e., acetylation and epimerization. Due to the critical role of c-di-GMP in the regulation of many biological processes, particularly the motility-sessility switch and also the emergence of persisting mucoid phenotypes, these results aid to reach a better understanding of biofilm-associated regulatory networks and c-di-GMP signaling and might assist the development of inhibitory drugs.
Bacterial biosynthesis of alginates Hay, Iain D; Ur Rehman, Zahid; Ghafoor, Aamir ...
Journal of chemical technology and biotechnology (1986),
June 2010, Volume:
85, Issue:
6
Journal Article
Peer reviewed
Alginates are polysaccharides with many industrial and medical uses, from food additives to encapsulation agents in the emerging transplantation technologies. Alginate is composed of variable ...proportions of β-D-mannuronic acid and α-L-guluronic acid linked by 1-4 glycosidic bonds. Traditionally, commercial alginate has been produced by farmed brown seaweeds, but this alginate suffers from heterogeneity in composition and quality partly due to environmental variation. Two bacterial genera, Pseudomonas and Azotobacter, are also capable of producing alginate as an exopolysaccharide. These bacterial alginate producers can provide the means to produce alginates with defined monomer composition and possibly through genetic and protein engineering may allow for the production of 'tailor made' bacterial alginates. The paper discusses the mechanisms behind alginate production in bacteria and how they may be used in the commercial production of alginates. Copyright
Polyhydroxyalkanoates (PHAs) are naturally occurring organic polyesters that are of interest for industrial and biomedical applications. These polymers are synthesized by most bacteria in times of ...unbalanced nutrient availability from a variety of substrates and they are deposited intracellularly as insoluble spherical inclusions or PHA granules. The granules consist of a polyester core, surrounded by a boundary layer with embedded or attached proteins that include the PHA synthase, phasins, depolymerizing enzymes, and regulatory proteins. Apart from ongoing industrial interest in the material PHA, more recently there has also been increasing interest in applications of the PHA granules as nano-/micro-beads after it was conceived that fusions to the granule associated proteins (GAPs) provide a way to immobilize target proteins at the granule surface. This review gives an overview of PHA granules in general, including biogenesis and GAPs, and focuses on their potential use as nano-/micro-beads in biotechnological and biomedical applications.
Recombinant protein production and purification from Escherichia coli is often accompanied with expensive and complicated procedures, especially for therapeutic proteins. Here it was demonstrated ...that, by using an intein cleavable polyhydroxyalkanoate synthase fusion, recombinant proteins can be first produced and sequestered on a natural resin, the polyhydroxyalkanoate (PHA) inclusions, then separated from contaminating host proteins via simple PHA bead isolation steps, and finally purified by specific release into the soluble fraction induced by a pH reduction.
By translationally fusing a target protein to PHA synthase using a self-cleaving intein as linker, intracellular production of PHA beads was achieved. Upon isolation of respective PHA beads the soluble pure target protein was released by a simple pH shift to 6. The utility of this approach was exemplified by producing six target proteins, including Aequorea victoria green fluorescent protein (GFP), Mycobacterium tuberculosis vaccine candidate Rv1626, the immunoglobulin G (IgG) binding ZZ domain of protein A derived from Staphylococcus aureus, human tumor necrosis factor alpha (TNFα), human granulocyte colony-stimulating factor (G-CSF), and human interferon alpha 2b (IFNα2b).
Here a new method for production and purification of a tag-less protein was developed through intein cleavable polyhydroxyalkanoate synthase fusion. Pure target protein could be easily obtained without laborious downstream processing.
Neoantigen-based cancer vaccines have emerged as a promising immunotherapeutic approach to treat cancer. Nevertheless, the high degree of heterogeneity in tumors poses a significant hurdle for ...developing a vaccine that targets the therapeutically relevant neoantigens capable of effectively stimulating an immune response as each tumor contains numerous unique putative neoantigens. Understanding the complexities of tumor heterogeneity is crucial for the development of personalized neoantigen-based vaccines, which hold the potential to revolutionize cancer treatment and improve patient outcomes. In this review, we discuss recent advancements in the design of neoantigen-based cancer vaccines emphasizing the identification, validation, formulation, and targeting of neoantigens while addressing the challenges posed by tumor heterogeneity. The review highlights the application of cutting-edge approaches, such as single-cell sequencing and artificial intelligence to identify immunogenic neoantigens, while outlining current limitations and proposing future research directions to develop effective neoantigen-based vaccines.
The supermucoid Pseudomonas aeruginosa strain PDO300Δalg8(pBBR1MCS-5:alg8) showed strongly impaired attachment compared with the respective mucoid or nonmucoid strains and formed a thicker biofilm ...with large extended mushroom-like microcolonies. Alginate lyase treatment dissolved microcolonies. The data suggested that alginate overproduction impairs attachment but plays a structural role in microcolony formation.
Here, the class I polyhydroxyalkanoate synthase (PhaC) from Ralstonia eutropha was investigated regarding the functionality of its conserved C-terminal region and its ability to tolerate ...translational fusions to its C terminus. MalE, the maltose binding protein, and green fluorescent protein (GFP) were considered reporter proteins to be translationally fused to the C terminus. Interestingly, PhaC remained active only when a linker was inserted between PhaC and MalE, whereas MalE was not functional. However, the extension of the PhaC N terminus by 458 amino acid residues was required to achieve a functionality of MalE. These data suggested a positive interaction of the extended N terminus with the C terminus. To assess whether a linker and/or N-terminal extension is generally required for a functional C-terminal fusion, GFP was fused to the C terminus of PhaC. Both fusion partners were active without the requirement of a linker and/or N-terminal extension. A further reporter protein, the immunoglobulin G binding ZZ domain of protein A, was translationally fused to the N terminus of the fusion protein PhaC-GFP and resulted in a tripartite fusion protein mediating the production of polyester granules displaying two functional protein domains.
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