Creative Biolabs develops and commercializes a full range of integrated innovative services that are based on phage display technology. We have ...
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1. What is the surface display technology?[Top]
Surface display technology indicates expressing the exogenous proteins/peptides on the surface of the host (from simplest virus to mammalian cells) by fusing the exogenous genes with membrane proteins genes or modifying proteins/peptide to be anchored by host surface elements. The principal advantage of this technology is that it provides an efficient approach to link the genotype and phenotype. By saving the protein purification process, it facilitates the research of proteins/peptides affinity for its targets and protein functions as enzymes. In practical, surface display is quite a useful tool in drug discovery by high throughput screening for the proteins/peptides binding to specific ligand and blocking their activities.
Phage or bacteriophage is a kind of virus with DNA or RNA encapsulated by coat proteins. They can infect bacteria as a host to insert their genes into bacteria genome and produce coat proteins and genes to reform phage particles in cytoplasma followed by secretion into periplasm. The plasmid that is able to produce phages after transformation into bacteria is called phagemid. When exogenous genes are inserted into phagemid and transform into bacteria, phages with exogenous proteins/peptides displayed on the capsid will be produced.
Since the advent of phage display technology in 1985 by G. Smith and the first collection of antibody libraries displayed on phages were published in 1990, phage display has been widely used in protein/antibody characterization and most importantly, antibody discovery. Conventional approach to generate antibodies requires immunization of host animals and subsequent complicated purification steps. In addition, the antibodies in clinical applications must be human origin and it is apparently not practical to immunize human with certain antigen, so downstream sequencing and humanization of animal origin antibodies are required, which are usually costly and bring unpredictable problems to the antibodies. Furthermore, the traditional approach needs myeloma cells of the host, which is not always available for animals other than mice. In contrary, phage display based antibodies screening can circumvent these problems, but require the establishment of antibody libraries. The sources of the libraries include nonimmunized human B cells, antigen immunized human or animal B cells, in vitro gene synthesis by randomly combination of various VDJ genes of human. After the gene pool has been built from above sources, it is subcloned into phage display vectors and then transform into appropriate bacteria to harvest the phage libraries. Afterwards, phages library is subject to several round of affinity panning for collecting the specific antigen binding antibodies displayed on phage. Then, the monoclonal antibodies were identified by ELISA, sequenced and cloned into mammalian cells for large scale production.
Briefly, the yeast display indicates exogenous proteins/peptides are expressed on cell surface by linked or anchored to yeast cell wall composition. To achieve this, exogenous genes are fused with cell wall proteins genes, of which Aga2p is the most commonly used for antibodies display. Aga2p, belonging to yeast agglutinins protein family responsible for mating events, is anchored to cell surface via two disulfide bonds to Aga1p. One advantage for utilizing Aga2p as the fusion protein is that it is relative far from the cell wall, so the fused antibodies are more flexible in space, avoiding the loss of activity due to steric hindrance. The other advantage is that Aga2p is expressed after cell growth under the control of GAL1 promoter, protecting yeast cells from potential toxic antibody thus ensuring the display of all the antibodies in the library. Yeast with antibodies or other proteins displaying is then subject to screening with antigen coated magnetic beads, followed by several rounds of FACS screening for the antibody/proteins with desirable properties.
Apart from the application in antibody discovery, yeast display is widely used in the fermentation industry. Yeast cells with whole enzyme or the activity site of enzyme are involved in the fermentation system. When compared to traditional methods using purified enzymes, yeast display aided fermentation has advantages like reducing enzyme cost, easily control of the fermentation procedure and simplifying the downstream purification step.
4. What is the difference between yeast display and phage display?[Top]
Yeast display has advantages over phage display in some aspects, while it also has some drawbacks.
(1) In the screening/panning step, yeast display is suitable for FACS, allowing to sort out antibodies with desired binding properties precisely. However, the affinity of antibodies to antigens in phage display can only be roughly stratified by adjusting the washing buffer compositions.
(2) Owing to its eukaryotic expression system, yeast displayed antibodies can be correctly folded and modified, such as glycosylation, to be closer to their native structure in mammals compared to the antibodies expressed by E.coli in phage display.
(3) The diversity of yeast displayed antibodies may be lower than phage (107-109 for yeast, up to 1011 for phage), due to the low transformation efficiency of yeast.
(4) Due to the much more copies of antibody on yeast surface (104-105) than phage, it is possible to select out low affinity antibodies according to antibody avidity with yeast display system.
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