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Bio-fishing Platform
Creative Biolabs develops and commercializes a full range of integrated innovative Bio-fishing services that are based on phage display technology. We have extensive experience in construction of various phage display libraries, including cDNA, peptide and scFv/Fab antibody libraries. We are also professional in screening these libraries against protein, peptide, cells, special surfaces and chemicals. Affinity maturation based on sub-library construction and screening is readily available. In addition, we have built up a series of peptide, human and mouse scFv/Fab phage display libraries that all have a great diversity. We also have a camelised human single domain antibody library constructed. Furthermore, bio-manufacturing of small antibodies in bacterial systems and production of full-size humanized IgG in gene-engineered CHO cells have brought us international recognition. Our Bio-fishing platform enables rapid and effective discovery and efficient production of biological molecules that can provide opportunities for therapeutics, diagnostics and targeting agents. Introduction to Phage Display Technology In a phage display library, a variety of peptides, small antibodies [e.g. scFv and Fab] or proteins are displayed on the surface of filamentous phage [M13, fd, and f1 strains] and lambda phages as fusion proteins with one of the coat proteins of the phage virions, while the genetic materials encoding the proteins are housed within the virion. Using a binding affinity-based process called panning [Figure 1], a small number of phages that display proteins specifically binding to a target of interest can be rescued from a phage library that usually has a repertoire of many billions of unique displayed proteins. Finally, the proteins displayed by the selected phages can be identified by phage amplification and DNA sequencing.
Fig. 1. Illustration of phage display cycle.
Phage display systems can be grouped into two classes based on the vector system. o True phage vector system - Phage vectors are usually derived from the genome of filamentous phage (M13, f1, or fd), encoding all proteins needed for the replication and assembly of the filamentous phage. - The library is ether cloned as a fusion with the coat protein originally present in the phage genome or inserted as fusion gene cassette with an additional copy of the coat protein yielding phages that present the wild type and the fusion coat protein on the same phage particle. o Phagemid system - Phagemid is a plasmid that bears a phage-derived origin of replication in addition to its plasmid origin of replication. The phage-derived origin of replication is also known as intergenic region, functioning in DNA replication and the packaging of the ssDNA in the phage coat. o Phages containing the phagemid genome can be produced in the presence of phage-derived proteins, which are simply provided by super-infecting phagemid-carrying cells with a helper phage. - Two distinct types of phage particles are produced carrying either the phagemid genome or the helper phage genome. The latter ones can be reduced by using a helper phage with a defective origin of replication or packaging signal to preferentially package phagemid DNA over the helper phage genome.
- Phagemid-based display systems usually yield phages with a hybrid phenotype displaying wild type and fusion coat protein on the same particle.
Categorization of Phage Display Libraries Phage display peptide libraries made with synthetic oligonucleotides are efficient in identifying peptides that interact with many different kinds of bait ligands, including proteins, peptides, RNAs, and oligonucleotides. Peptide libraries are also a good tool to identify substrate cleavage sites of various proteases. Most importantly, phage display scFv and Fab antibody library screening allows production of specific antibodies targeting any possible antigens, including self-antigens, without prior immunization. Phage display cDNA libraries provide an alternative research approach for protein-protein interaction, which is complementary to yeast two-hybrid system. Besides, phage display libraries have been used in vivo for selection of tissue-targeting proteins. o Combinatorial peptide library - Flexible and constrained random peptide library can be constructed using either of the above two phage display systems for screening peptides with different binding potential. o Antibody library - Format: Effective display formats include scFv, Fabs, and single variable chain (VH or VL). - Gene resource: Antibody genes are cloned from immunized animals/immune donors, non-immunized donors or synthesized by introducing a predetermined level of randomization into complementary determining regions (CDR). - Application: Selection of new antibodies against purified antigen, complex antigen, and intact cells; in vivo selection; target discovery and validation; selection of antibodies with improved stability, folding and other properties; selection for a particular function. o cDNA library In the majority of phage display systems, displayed proteins are expressed as a C-terminal fusion with the N-terminus of the minor coat protein. Since full-length cDNAs generally contain several stop codons near the 3’ end; thus full-length proteins cannot be displayed on the phage surface using these systems. Recent development in the field has resolved the issue. - Protein domain cDNA library: By displaying protein fragments instead of full-length cDNAs, protein domain libraries can be constructed in phage display formats. - Jun/Fos pseudo-fusion phage display system: a fusion of Fos leucine zipper domain to the 5'-end of a cDNA library is captured by a fusion of Jun leucine zipper domain to the N terminus of pIII coat protein. o pVI fusion phage display system: A cDNA library is fused to the C-terminus of the phage protein pVI. - Display on the surface of lytic bacteriophage: cDNA encoded proteins are fused at the C-terminal end of a capsid protein (Lambda phage or T7 phage) or a coat protein (T4 phage) in these novel phage display systems.
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