Ribosome complexes as selection particles for in vitro display and evolution of proteins

A method capable of (I) displaying and selecting proteins and peptides and (II) recovering genetic material selected from the group consisting of DNA and mRNA that encodes the proteins or peptides, wherein the method comprises:

• (a) conducting coupled transcription of DNA lacking a stop codon and translating mRNA therefrom in a cell free rabbit reticulocyte system in order to from complexed particles comprising at least one individual nascent protein or peptide associated with (i) one or more ribosomes and (ii) the mRNA encoding the protein or peptide;
• (b) contacting the complexed particles with an immobilized ligand, antigne or antibody;
• (c) selecting complexed particles using binding of the ligand, antigen or antibody to the protein or peptide of the complexed particle;
• and
• (d) conducting reverse transcription and polymerase chain reaction (RT-PCR) on the mRNA bound in the selected complexed particle from step (c) in order produce DNA encoding the protein or peptide.

A method capable of (I) display proteins and peptides and (II) recovering genetic material selected from the group consisting of DNA and mRNA that encodes the proteins or peptides, wherein the method comprises:

• (a) translating an mRNA or an mRNA library in a eukaryotic cell free system in order to form complexed particles, wherein a complexed particle comprises at least one individual nascent protein or peptide associated with (i) one or more ribosomes and (ii) the RNA encoding the protein or peptide;
• (b) contacting the particles with a ligand, antigen or antibody;
• (c) selecting complexed particles using binding of the ligand, antigen or antibody with the protein or peptide of the complexed particle;
• and
• (d) conducting reverse transcription and polymerase chain reaction (RT-PCR) on the mRNA bound in the selected complexed particle in order to produce DNA encoding the protein or peptide.

A method capable of (I) displaying proteins and peptides and (II) recovering genetic material selected from the group consisting of DNA and mRNA that encodes the proteins or peptides, wherein the method comprises:

• (a) transcribing a cDNA or a cDNA library and translating mRNA therefrom in a eukaryotic cell free system in order to form complexed particles, wherein a complexed particle comprises at least one individual nascent protein or peptide associated with (i) one or more ribosomes and (ii) the mRNA encoding the protein or peptide;
• (b) contacting the particles with a ligand, antigen or antibody;
• (c) selecting complexed particles using binding of the ligand, antigen or antibody with the protein or peptide of the complexed particle;
• and
• (d) conducting reverse transcription and polymerase chain reaction (RT-PCR) on the mRNA bound in the selected complexed particle from step (c) in order to produce DNA encoding the protein or peptide.

A method of displaying and selecting proteins and peptides and recovering genetic material that encodes the proteins or peptides, wherein the method comprises:

• (a) transcribing and translating DNA in a cell free system in order to form complexed particles, wherein a complexed particle comprises at least one individual nascent protein or peptide associated with (i) a ribosome and (ii) the mRNA encoding the protein or peptide;
• (b) contacting the complexed particles with a ligand, antigen or antibody;
• (c) selecting complexed particles using binding of the ligand, antigen or antibody with the protein or peptide of the complexed particle;
• (d) conducting reverse transcription and polymerase chain reaction (RT-PCR) on the mRNA bound in the selected complexed particle from step (c) in order produce DNA encoding the protein or peptide;
• and
• (e) recovering the DNA encoding the protein or peptide.

A method for making mammalian antibodies, wherein the method comprises:

• (a) contacting an animal with an antigen,
• (b) making a DNA library comprising combinations of the VH and VL regions of the immunoglobulins of said animal, wherein the regions are linked as single chain Fv or VH/K fragments,
• (c) creating a eukaryotic ribosome display library by in vitro transcription of said DNA library and in vitro translation of mRNA therefrom, such that complexed particles are formed, wherein a complexed particle comprises at least one individual nascent antibody, fragment associated with (i) one or more ribosomes and (ii) the mRNA encoding the antibody fragments,
• (d) selecting complexed particles carrying specific antibody fragments through binding to an antigen or other agent,
• (e) conducting reverse transcription and polymerase chain reaction (RT-PCR) on the mRNA bound in the selected complexed particle from step (d) in order to produce DNA encoding the protein or peptide, and
• f) recovering and expressing the DNA from step (e).

A method for making human antibodies, wherein the method comprises (a) contacting with an antigen a transgenic mouse carrying human loci encoding heavy and/or light chains of immunoglobulins as transgenes,

• (b) making a DNA library comprising combinations of the VH and VL regions of the human immunoglobulins from the mouse, wherein the regions are linked as single chain Fv or VH/K fragments,
• (c) creating a eukaryotic ribosome display library by in vitro transcription of said DNA library and in vitro translation of mRNA therefrom, such that complexed particles are formed, wherein a complexed particle comprises at least one individual nascent antibody fragment associated with (i) one or more ribosome and (ii) the mRNA encoding the antibody fragment,
• (d) selecting such complexed particles carrying specific antibody fragments through binding to an antigen or other agent,
• (e) conducting reverse transcription and polymerase chain reaction (RT-PCR) on the mRNA bound in the selected complexed particle from step (d) in order to produce DNA encoding the protein or peptide, and
• f) recovering and expressing the DNA from step (e).