Applications of Microbial Biopolymers in Display Technology
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Microorganisms produce a variety of different polymers such as polyamides, polysaccharides, and polyesters. The polyesters, the polyhydroxyalkanoates (PHAs), are the most extensively studied polymers in regard to their use in display technology. The material properties of bacterial PHAs in combination with their biocompatibility and biodegradability make them attractive substrates for use in display technology applications. By translationally fusing bioactive molecules to a gene encoding a PHA-binding domain, the appropriate functionalization for a given application can be achieved such that the need for chemical immobilization is circumvented. By separately extracting and processing the biopolymer, using it to coat a surface, and then treating this surface with the fusion proteins, surface functionalization for immunodiagnostic microarray or tissue engineering applications can be accomplished. Conversely, by expressing the fusion protein directly in the PHA-producing organisms, one-step production of functionalized beads can be achieved. Such beads have been demonstrated in diverse applications, including fluorescence-activated cell sorting, enzyme-linked immunosorbent assays, microarrays, diagnostic skin test for tuberculosis, vaccines, protein purification, and affinity bioseparation.
3 Microarray Applications of Polyhydroxyalkanoates
The PHA material properties enable film coating of solid surfaces suitable for microarray applications . Indeed, by exploiting the ability of PHA-associated proteins to specifically bind PHAs, and thereby overcoming specificity- or orientation-associated issues, PHA substrates for immobilization have been demonstrated as attractive for such applications.
The first relevant example of PHA as a protein micropatterning substrate was described in a study by Park and colleagues (2005). Poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) were independently produced, purified, and used to spin-coat glass substrate, producing PHA films. Subsequently, enhanced green fluorescent protein and red fluorescent protein, fused with the hydrophobic side chain-interacting substrate binding domain (SBD) of a PHA depolymerase , were micropatterned onto the film using microcontact printing. Confocal fluorescent microscopy clearly revealed the printed patterns after several wash steps and surface plasmon resonance spectroscopy using anti-green fluorescent protein polyclonal antibody further confirmed this specific fusion protein immobilization, additionally demonstrating the system as capable for examining protein-protein interactions.
Spurred on by these encouraging results, a further study aimed to demonstrate the possibilities of this system for protein microarray development centered on immunodiagnostic applications (Park et al. 2006). PHA depolymerase SBD fusions with the single-chain antibody variable region (scFv) against hepatitis B virus (HBV) preS2 surface protein as well as the severe acute respiratory syndrome coronavirus envelope protein (SCVe) were produced and microspotted onto P(3HB) films. Fluorescence-labelled HBV antigen and anti-SCVe antibody were then used to detect the interactions on the films . Fluorescence signals were detected only at the corresponding microspotted regions, indicating high affinity and selectivity and thus indicating the technology as appropriate for use in immunodiagnostics.
4 Tissue Culture/Engineering Applications of Polyhydroxyalkanoates
The diverse, adjustable material properties of PHAs in concert with their biodegradability , biocompatibility , noncarcinogenicity, and low cytotoxicity have driven their development in the area of biomaterials research (Ali and Jamil 2016). On the basis of PHA surface functionalization by addition of PHA-binding protein fusions, several approaches to enhancing tissue engineering and culturing techniques have been undertaken.
By translationally fusing the PHA-binding phasin, PhaP, to the cell adhesion motif Arg-Gly-Asp (RGD) and applying this fusion protein to coat PHA surfaces, the effect on fibroblast growth was investigated (Dong et al. 2010). Under serum-free conditions, confocal laser scanning microscopy in combination with cell counting assays revealed a significant cell attachment increase on the PhaP-RGD-coated PHA films relative to the PhaP- (nonfused) and noncoated films, in a manner that is unlikely to be attributable to changes in PHA surface topology. Further cell proliferation assays revealed increased fibroblast proliferation levels on PhaP-RGD-coated PHA films. Thus, PHA surface functionalization, using a well-known cell adhesion motif, was demonstrated using aqueous solutions, avoiding toxicity of chemical immobilization techniques. Continuing this pattern of investigation, similar increases in cell adhesion and cell proliferation of human vascular smooth muscle cells (HvSMCs) were demonstrated using the PHA repressor protein (PhaR) fused to the specific integrin ligand peptide (KQAGDV) (Dong et al. 2012), and, more recently, PhaP fused to RGD or the laminin-derived IKVAV peptides used to coat PHA films demonstrated enhanced neural stem cell (NSC) attachment, proliferation, and better neurite outgrowths, without effecting NSC differentiation (Xie et al. 2013).
5 Diagnostic Applications: In Vitro and In Vivo
5.1 In Vitro Diagnostics
Fluorescence activated cell sorting (FACS) is a qualitative and quantitative technique used in biomolecule detection for in vitro diagnostics. Generally, antigen-displaying beads are used to bind antibodies which are then detected via fluorescent-signaling secondary antibodies when the bead suspension passes through the FACS machine flow cell. Current bead preparation techniques involve tedious antigen purification and chemical crosslinking. Therefore, more cost-effective, reliable means of producing antigen-displaying beads with comparable detection efficacy could greatly impact the accessibility of FACS. A series of studies have shown that one path towards such improvement may be via one-step production of surface protein engineered PHA beads.
The enzyme-linked immunosorbent assay (ELISA) is based on antigen-antibody binding on solid support either detecting antigen or antibody in samples. In vivo assembled engineered PHA inclusions displaying antigens or specific binding domains as described above were directly used to coat ELISA plates. Respective ELISAs showed specific and sensitive detection of the corresponding antibody or antigen confirming the applicability of bioengineered PHA beads in ELISA (Peters and Rehm 2008; Atwood and Rehm 2009; Parlane et al. 2009).
5.2 Skin Test
One in vivo-produced PHA bead-based approach to in vivo diagnosis is a skin test for the detection of bovine tuberculosis (TB) (Chen et al. 2014). The major disadvantage of the currently widespread tuberculin skin test is the lack of detection specificity for pathogenic Mycobacterium tuberculosis complex members, that is, cattle exposed to nonpathogenic, environmental mycobacteria may result in false-positives. Thus, with the aims of improving specificity and lowering production cost, an alternative test was developed. Three immunodominant TB antigens, which are absent in most environmental mycobacteria, were fused to PhaC mediating production of triple-antigen-displaying PHA beads. These granules showed in vitro increased reactivity with antigen-specific TB antibodies when compared with granules displaying only one antigen. Assessment of triple-antigen-displaying PHA beads in the skin test (in vivo) showed specificity as all cattle experimentally infected with Mycobacterium bovis were detected while no false-positive reactions in cattle previously exposed to environmental mycobacteria were observed. A fourth antigen was added to the triple-antigen-displaying PHA beads to boost skin test sensitivity (Parlane et al. 2016). Dose response studies showed that very low amounts of mycobacterial antigens (≥0.1 μg) were already sufficient for the skin test suggesting greatly increased immunogenicity of antigen-displaying beads versus soluble antigens. Hence, it was demonstrated that antigen-displaying PHA beads mediate an antigen-specific immune reaction upon injection into the skin, that is, serve as specific immune response stimulating antigen delivery systems .
6 Particulate Antigen Delivery Systems
7 Recombinant Protein Production via Display on Polyhydroxyalkanoate Inclusions
Purification of recombinantly produced proteins, generally with the target protein fused to an affinity tag, can be a time-consuming and costly process since it often requires multiple chromatography steps which all need to be individually optimized, and additionally require cleavage and removal of the tag. Over the past 10 years, various approaches have been undertaken to make use of PHA and PHA-associated proteins for the development of alternative, simple and cost-effective protein purification systems. The general principle that most of these studies have followed is to produce the target protein as a translational fusion to a PHA inclusion-associated protein in cells that also make PHA and to then copurify the protein with the PHA beads. This can be done in a host that naturally produces PHA or in an organism that has been genetically engineered to make PHA (e.g., E. coli). Different methods have then been applied to release the target protein from the beads. Early examples used the phasin protein PhaP as an affinity tag and inducible self-cleaving inteins inserted between PhaP and the target protein to release the protein of interest. This method was applied by Barnard and coworkers to purify green-fluorescent protein (GFP) and β-galactosidase (LacZ) from Ralstonia eutropha, a natural PHB producer (Barnard et al. 2005). GFP and LacZ could be successfully purified fused to either end of PhaP using a thiol-inducible intein. Similarly, several proteins (maltose-binding protein (MBP), LacZ, chloramphenicol acetyltransferase (CAT), and NusA) were tagged with PhaP and purified from recombinant E. coli with the help of a pH-inducible intein (Banki et al. 2005).
To strengthen the binding of the fusion protein to the bead surface to the point where significant leakage during the purification process could be avoided, multiple (two or three) phasins were used as a tag. Geng and coworkers chose a protein which is generally difficult to produce in bacteria due to the presence of several disulfide bonds (Geng et al. 2010). They fused recombinant human tissue plasminogen activator (rPA), a truncated version of tissue plasminogen activator, to PhaP and inserted a thrombin cleavage site as a linker. Active rPA could be released from isolated beads by thrombin treatment.
In another study which focused on diagnostic applications (aforementioned) the MOG and interleukin-2 were produced as PhaP-fusions containing an enterokinase recognition site (Bäckström et al. 2007). The successful removal of MOG or IL2, respectively, after enterokinase treatment was monitored by FACS analysis.
A slightly different approach to PHA-based protein purification is based on production of fusion proteins separate to PHA extraction and processing into beads. Various target proteins were fused to different granule-associated proteins such as PhaP or the regulatory protein PhaR, the fusion proteins recombinantly produced in E. coli and crude cell lysates incubated with beads processed from extracted PHA (Wang et al. 2008, Zhang et al. 2010). Release of the target protein was also achieved via intein-mediated self-cleavage. While this method is more labor-intensive than producing PHA and proteins in the same cell, advantageous might be the possibility to produce the tagged target protein in any host organism including eukaryotes. A simplified approach used the N-terminal part of PhaF to anchor a target protein to PHA beads in vivo, followed by bead isolation and release of the target protein (i.e., the entire fusion of target protein and PhaP) from the beads by detergent treatment (Moldes et al. 2004).
However, a general drawback of using PhaP (or PhaR) as a tag for protein purification is that these proteins are only attached to the PHA inclusion surface via hydrophobic interactions, so there is a risk that the fusion protein could detach during either the bead purification process or the tag cleavage process resulting in loss of target protein. In the natural system, phasins have the advantage of being the predominant protein on the surface of PHA inclusions (Wieczorek et al. 1995); however, in a recombinant system, proteins such as the PHA synthase can be overproduced to achieve a high density at the inclusion surface (Brockelbank et al. 2006; Mifune et al. 2009).
Grage et al. harnessed the covalent attachment of the PHA synthase to PHA inclusions by translationally fusing HcRed or an anti-β-galactosidase single-chain antibody fragment (scFv) (Martineau et al. 1998), separated by an enterokinase recognition site (Grage et al. 2011). Both target proteins were successfully released from purified beads by enterokinase treatment; however, cleavage efficiency was relatively low. In an attempt to find a robust and inexpensive auto-processing module, Hay and coworkers used a modified soluble form of the cell surface sortase transpeptidase A (SrtA) from Staphylococcus aureus which had been engineered to self-cleave in the presence of Ca2+ (Mao 2004; Hay et al. 2015a). SrtA and the target protein were fused to the C-terminus of PhaC using an extended linker (Jahns and Rehm 2009). Using this technique, GFP, MBP, and antigen RV1626 from Mycobacterium tuberculosis could be released from isolated PHA beads at a high yield and purity (e.g., 6 mg/l of soluble GFP at a purity of about 98%) (Hay et al. 2015a).
8 Affinity Bioseparation Applications Using Display of Binding Domains
It may not always be feasible or desirable to produce the target protein fused to a bead-associated protein (and to coproduce it with the beads/resin). Hence PHA beads have also been engineered to serve as affinity resins for bioseparation, generally exploiting the possibility of densely displaying binding domains on nano-/microbeads . These resins can be produced in one step by fusing the binding domain of choice to the bead-associated protein and then isolating these functionalized beads from the production host (similar to the protein purification approach described above). The first example of this was the immunoglobulin G (IgG) binding ZZ domain of Staphylococcus aureus protein A translationally fused to PhaC (Brockelbank et al. 2006). The resulting ZZ domain displaying beads were isolated from E. coli and successfully purified IgG from human serum and mouse hybridoma supernatants, with purity and yield comparable to commercially available protein A sepharose (Brockelbank et al. 2006; Lewis and Rehm 2009).
Further PHA bead-based resins assembled by engineering PhaC displayed streptavidin which bound various biotinylated compounds such as enzymes, antibodies, and DNA (Peters and Rehm 2008). Grage and Rehm were able to purify β-galactosidase from a mixture of proteins using anti-LacZ scFv immobilized to PHA beads (Grage and Rehm 2008). An endotoxin-removing resin was developed by producing human lipopolysaccharide-binding protein (hLBP) fused to PhaP in Pichia pastoris (Li et al. 2011). After secretion and recovery from the culture supernatant, the hLBP-PhaP fusion was incubated with beads processed from extracted PHA, and the resulting hLBP-beads were tested for their endotoxin removal abilities under a variety of conditions. According to the authors, the beads performed better than commercially available endotoxin-removing gels (Li et al. 2011).
Recently, Hay and coworkers published a more extensive study that aimed at broadening the applicability of PHA bead affinity resins by identifying and testing several easily customizable affinity binding domains which they translationally fused to PhaC (Hay et al. 2015b). This study demonstrated that VHH domains from camelid antibodies , designed ankyrin repeat proteins (DARPins) and OB-folds (OBodies), could be densely displayed on PHA beads resulting in high affinity binding resins for purification of various target proteins. These binding domains were used to establish extensive libraries of variants enabling screening for binders specific for the target compound of interest (Binz et al. 2004; Harmsen and De Haard 2007; Stumpp et al. 2008; Steemson et al. 2014). PHA-based affinity resins showed a purification performance at least equal to current commercial offerings (Hay et al. 2015b). A recent surface topology study of the R. eutropha PhaC attached to PHA inclusions suggested new engineering strategies towards the development of PHA-based affinity resins with increased binding capacity via improved display (Hooks and Rehm 2015). This study identified several surface-exposed flexible regions of PhaC, which tolerated the insertion of the IgG-binding ZZ domain. One of the double ZZ domain insertions (i.e., ZZ inserted in two of the surface-exposed regions) showed greatly improved IgG binding capacity with some of the single insertions also showing improved IgG binding capacity when compared with terminal fusions. Overall, PhaC engineering such as N- or C-terminal fusions and/or insertions enabled efficient display of binding domains for interaction with target compounds resulting in purification performance suitable for application as bioseparation resin.
9 Research Needs
Microbial PHAs show great promise as polymers providing a support structure for display of a range of protein functions. Since PHAs can be composed of various constituent resulting in a diversity of material properties, it currently remains unexplored how these different PHAs perform in the context of anchoring binding domains for display. Additionally, research needs to address bioprocessing challenges to obtain PHAs of consistent structure and shape for improved implementation in various protein display applications.
10 Concluding Remarks
Although microorganisms are capable of producing a variety of polymers, the hydrophobic thermoplastic PHAs currently hold the greatest promise as support material to display protein functions. The PHA material properties allow processing into nano-/microbeads, films, and 3D structures, while PHA-related proteins provide specific PHA binding domains to anchor protein functions of interest. These approaches enabled implementation in microarray-based diagnostics as well as tissue engineering. Besides the binding of protein functions to isolated and processed PHA, recent research elucidated the concept of producing PHA inclusions within the bacterial cell already coated with desired protein functions. The applicability of these PHA beads as vaccines, in diagnostics and as bioseparation resin was demonstrated.
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