Antibody Libraries and Phage Display

Nowadays, phage display in combination with antibody gene libraries is widely used to select Escherichia coli host cells that express desired antibody fragments. Such gene libraries are typically produced either from natural sources (e.g., from the spleen of an immunized animal or from plasma cells of human donors) or generated by genetic engineering. The latter has been used to create naive libraries based on one or more antibody VH and VL gene segments that are diversified by cassette mutagenesis or similar approaches. Such libraries are typically unbiased and can be used for any given antigen (1-4). Modern naive libraries are generally large (more than 1010 members), contain only few nonfunctional members, yield antibodies that are well expressed in E. coli (more than 1 mg/L of purified material), and are designed to allow further affinity maturation, if needed. Phage display is then most often used to select desired antibodies from such libraries (for reviews, see refs. 5 and 6). First, E. coli host cells are transformed with the library that has been inserted into phagemid vectors bearing the antibody gene fragments fused to the phage gene III that will later be part of the phage particle. Host cells carrying the phagemid are selected with an antibiotic-resistance marker. For phage display, the culture is infected with a helper phage that will deliver all necessary components for phage production. During infection, the cloned antibody sequences are transcribed, and the sequences are produced as a fusion protein, consisting of a bacterial "leader sequence," the antibody gene fragment(s), and the gill phage coat protein. The leader sequence directs the proteins to the periplasmic space, where the fused antibody fragment is incorporated into viable phage particles via the coat protein. Phages are secreted through the bacterial outer membrane and display one to three copies of the encoded antibody fragment on their outer surfaces. Such phages can be easily isolated and stored for later use.

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  • ian
    Can helper phage be stored at 20?
    2 months ago

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