24 Diagnostic Applications of Phycobiliproteins
603
creation of protein conjugates, fusion proteins, adducts, linking of antibodies with
PBPs, preparation of micro beads and magnetic beads coated with PBPs, and linking
of these pigments with DNA probes. Patents pertaining to the development of such
methods are listed in the global context (Table 24.10). It primarily includes patents
from United States Patent and Trademark Office (USPTO) dealing with core methods
of developments followed by many patents from China National Intellectual Property
Administration (CNIPA), China.
The following types of preparations containing PBPs as detection component
were commonly encountered among them.
Recombinant fusion proteins
The recombinant fusion protein, streptavidin-phycobiliprotein conjugates was
produced in E. coli by combinational biosynthesis. There were two recombinant fusion proteins—SA-PCA-PEB (streptavidin-phycocyanin α subunitphycoerythrobilin), and SA-PCA-PCB (streptavidin-phycocyanin α subunitphycocyanobilin). Purification of these conjugates was efficient using biotinylated
substances due to the higher affinity of streptavidin to biotin. This combination
served the dual purpose of purification and fluorescence detection. These unnatural
recombinant phycobiliproteins were cross-linked products of phycobiliproteins with
phycobilins and exhibit improved fluorescence property (Ge et al. 2017). Another
recombinant fusion protein containing streptavidin and allophycocyanin α-subunit
(holo-ApcA) was biosynthesized in E. coli by a dual plasmid system. The rate of chromophorylation of the fusion protein with the prosthetic group phycocyanobilin (PCB)
was increased with an in vitro chromophore attachment reaction system containing
PCB and lyase gene cpcS yielding strong signal for detection (Wu et al. 2018). A
Similar system of fusion protein with tandem repeats of allophycocyanin holo-αsubunits to improve brightness of fluorescence has been carried out (Chen and Jiang
2018). Flow cytometric evaluation of coupled microspheres of anti-IgG-PE and antiHBsAg-PE conjugates was performed for quality control (de Almeida Santiago et al.
2016).
Magnetic beads
Preparations based on magnetic beads ease handling and purification using a
magnetic system in which the beads were coated with streptavidin. The shellfish
toxin, Okadaic acid was biotinylated which in turn binds to the magnetic beads
by the attraction of biotin to streptavidin. OA bind with the anti-OA monoclonal
antibodies which in turn bind with the secondary antibody IgG coupled with Rphycoerythrin. This preparation thus facilitates fluorescence detection (Pan et al.
2018). Such magnetic particle-based detection method and tool using phycoerythrin
was developed (Leigh et al. 2005).
Micro beads
Performance evaluation of polystyrene beads (0.86 μm) of phycoerythrin in terms
of quantity of antibodies bound per cell was made (Pannu et al. 2001).
603
creation of protein conjugates, fusion proteins, adducts, linking of antibodies with
PBPs, preparation of micro beads and magnetic beads coated with PBPs, and linking
of these pigments with DNA probes. Patents pertaining to the development of such
methods are listed in the global context (Table 24.10). It primarily includes patents
from United States Patent and Trademark Office (USPTO) dealing with core methods
of developments followed by many patents from China National Intellectual Property
Administration (CNIPA), China.
The following types of preparations containing PBPs as detection component
were commonly encountered among them.
Recombinant fusion proteins
The recombinant fusion protein, streptavidin-phycobiliprotein conjugates was
produced in E. coli by combinational biosynthesis. There were two recombinant fusion proteins—SA-PCA-PEB (streptavidin-phycocyanin α subunitphycoerythrobilin), and SA-PCA-PCB (streptavidin-phycocyanin α subunitphycocyanobilin). Purification of these conjugates was efficient using biotinylated
substances due to the higher affinity of streptavidin to biotin. This combination
served the dual purpose of purification and fluorescence detection. These unnatural
recombinant phycobiliproteins were cross-linked products of phycobiliproteins with
phycobilins and exhibit improved fluorescence property (Ge et al. 2017). Another
recombinant fusion protein containing streptavidin and allophycocyanin α-subunit
(holo-ApcA) was biosynthesized in E. coli by a dual plasmid system. The rate of chromophorylation of the fusion protein with the prosthetic group phycocyanobilin (PCB)
was increased with an in vitro chromophore attachment reaction system containing
PCB and lyase gene cpcS yielding strong signal for detection (Wu et al. 2018). A
Similar system of fusion protein with tandem repeats of allophycocyanin holo-αsubunits to improve brightness of fluorescence has been carried out (Chen and Jiang
2018). Flow cytometric evaluation of coupled microspheres of anti-IgG-PE and antiHBsAg-PE conjugates was performed for quality control (de Almeida Santiago et al.
2016).
Magnetic beads
Preparations based on magnetic beads ease handling and purification using a
magnetic system in which the beads were coated with streptavidin. The shellfish
toxin, Okadaic acid was biotinylated which in turn binds to the magnetic beads
by the attraction of biotin to streptavidin. OA bind with the anti-OA monoclonal
antibodies which in turn bind with the secondary antibody IgG coupled with Rphycoerythrin. This preparation thus facilitates fluorescence detection (Pan et al.
2018). Such magnetic particle-based detection method and tool using phycoerythrin
was developed (Leigh et al. 2005).
Micro beads
Performance evaluation of polystyrene beads (0.86 μm) of phycoerythrin in terms
of quantity of antibodies bound per cell was made (Pannu et al. 2001).