Compositions, methods, and kits are provided for efficiently generating and screening humanized antibody with high affinity against a specific antigen. The library of humanized antibody is generated by mutagenizing a chimeric antibody template that combines human antibody framework and antigen binding sites of a non-human antibody. Alternatively, the library of humanized antibody is generated by grafting essential antigen-recognition segment(s) such as CDRs of the non-human antibody into the corresponding position(s) of each member of a human antibody library. This library of humanized antibody is then screened for high affinity binding toward a specific antigen in vivo in organism such as yeast or in vitro using techniques such as ribosome display or mRNA display. The overall process can be efficiently performed in a high throughput and automated manner, thus mimicking the natural process of antibody affinity maturation.

A method for producing a ribosome display complex includes obtaining a ribosome complex including an unmodified polypeptide chain, an mRNA molecule and a ribosome by initiating translation of the mRNA molecule in a cell-free peptide synthesis system including the ribosome, and modifying the unmodified polypeptide chain by reacting a side chain reactive functional group in the unmodified polypeptide chain with a modifying reagent to produce a ribosome display complex including a modified polypeptide chain, the mRNA molecule and the ribosome. The unmodified polypeptide chain includes at least one reactive amino acid residue selected from the group consisting of a cysteine residue, a lysine residue, a histidine residue and a tryptophan residue. The at least one reactive amino acid residue includes the side chain reactive functional group, and the mRNA molecule includes a base sequence encoding an amino acid sequence of the polypeptide chain.

Compositions, methods and uses of high-diversity nucleic acid library that encodes a plurality of antibodies or antibody fragments are presented. The high-diversity nucleic acid library comprises or is derived from (1) a V.sub.H-CDR1/2 sub-library, (2) a plurality of V.sub.H-CDR3 sub-libraries, and (3) a V.sub.L sub-library, each of which comprises a plurality of members. Preferably, each member of the sub-libraries comprises at least one random cassette that has a plurality of degenerate base positions. In an especially preferred embodiment, at least portions of at least two members of the V.sub.H-CDR1/2 sub-library, the plurality of V.sub.H-CDR3 sub-libraries, and the V.sub.L sub-library are recombined to form an expression library member in an expression library, where each member of the expression library encodes a distinct antibody or antibody fragment.

Compositions, methods and uses of high-diversity nucleic acid library that encodes a plurality of antibodies or antibody fragments are presented. The high-diversity nucleic acid library comprises or is derived from (1) a V.sub.H-CDR1/2 sub-library, (2) a plurality of V.sub.H-CDR3 sub-libraries, and (3) a V.sub.L sub-library, each of which comprises a plurality of members. Preferably, each member of the sub-libraries comprises at least one random cassette that has a plurality of degenerate base positions. In an especially preferred embodiment, at least portions of at least two members of the V.sub.H-CDR1/2 sub-library, the plurality of V.sub.H-CDR3 sub-libraries, and the V.sub.L sub-library are recombined to form an expression library member in an expression library, where each member of the expression library encodes a distinct antibody or antibody fragment.

A binding agent to a target molecule, or method or kit where the binding agent is selected from a library where each variant has a circular permutation of the FHA domain where the rearrange does not substantially disrupt the FHA domain's beta-sheet scaffold or increase the stability of the beta-sheet scaffold. The randomized regions of the FHA domain include the endogenous binding interface the FHA domain, the region opposite of the endogenous binding interface, and the circular permutation region.

The invention provides methods for isolating cell-type specific mRNAs by selectively isolating ribosomes or proteins that bind mRNA in a cell type specific manner, and, thereby, the mRNA hound to the ribosomes or proteins that bind mRNA. Ribosomes, which are riboprotein complexes, bind mRNA that is being actively translated in cells. According to the methods of the invention, cells are engineered to express a molecularly tagged ribosomal protein or protein that binds mRNA by introducing into the cell a nucleic acid comprising a nucleotide sequence encoding a ribosomal protein or protein that binds mRNA fused to a nucleotide sequence encoding a peptide tag. The tagged ribosome or mRNA binding protein can then be isolated, along with the mRNA bound to the tagged ribosome or mRNA binding protein, and the mRNA isolated and further used for gene expression analysis. The methods of the invention facilitate the analysis and quantification of gene expression in the selected cell type present within a heterogeneous cell mixture, without the need to isolate the cells of that cell type as a preliminary step.

Methods for screening of affinity reagents for many target proteins of interest simultaneously. Arrayed targets (e.g., peptide, protein, RNA, cell, etc.) are used in affinity selection experiments to reduce the amount of target needed and to improve the throughput of discovering recombinant affinity reagents to a large collection of targets.

A combined ribosome-display and phage-display method and kit for carrying out the method are provided. The method includes screening a ribosome-display library of binding agents to identify binding agents that interact with one or more target molecules of interest, converting the RNA encoding the binding agents to a phage-display format by amplification and primer extension, and the screening the phage-display library to enrich for binding agents that interact with one or more target molecules of interest.

A recombinant Herpes Simplex Virus type 1 (HSV-1) strain H129-derived anterograde multi-synaptic transneuronal viral tracer for multi-synaptic neural circuit mapping comprises two or more fluorescence expression cassettes being integrated into the H129 genome at different locations; wherein each fluorescence expression cassette contains at least two copies of fluorescent protein-encoding sequence that are arranged in tandem, and at least one linker-encoding sequence, where at least one linker-encoding sequence is disposed between two fluorescent protein-encoding sequences, allowing transcription of fluorescent protein-encoding sequences and linker-encoding sequence as a single transcript; and wherein the linker-encoding sequence encodes a linker peptide containing at least two adjacent amino acids that are highly inefficient in forming a peptide bond between them; thereby, when the single transcript is translated, at least two fluorescent proteins are stoichiometrically generated due to the impedence of peptide bond formation by the linker peptide.

Methods for screening of affinity reagents for many target proteins of interest simultaneously. Arrayed targets (e.g., peptide, protein, RNA, cell, etc.) are used in affinity selection experiments to reduce the amount of target needed and to improve the throughput of discovering recombinant affinity reagents to a large collection of targets.