Abstract: The invention provides a method of displaying nascent proteins or peptides as complexes with eukaryotic ribosomes and the mRNA encoding the protein or peptide following transcription and translation in vitro, of further selecting complexes carrying a particular nascent protein or peptide by means of binding to a ligand, antigen or antibody, and of subsequently recovering the genetic information encoding the protein or peptide from the selected ribosome complex by reverse transcription and polymerase chain reaction (RT-PCR). The RT-PCR recovery step is carried out directly on the intact ribosome complex, without prior dissociation to release the mRNA, thus contributing to maximal efficiency and sensitivity. The steps of display, selection and recovery can be repeated in consecutive cycles. The method is exemplified using single-chain antibody constructs as antibody-ribosome-mRNA complexes (ARMs).
Abstract: Compositions, methods, and kits are provided for efficiently generating and screening a library of highly diverse protein complexes for their ability to bind to other proteins or oligonucleotide sequences. In one aspect of the invention, a library of expression vectors is provided for expressing the library of protein complexes. The library comprises a first nucleotide sequence encoding a first polypeptide subunit; and a second nucleotide sequence encoding a second polypeptide subunit. The first and second nucleotide sequences each independently varies within the library of expression vectors. In addition, the first and second polypeptide subunit are expressed as separate proteins which self-assemble to form a protein complex, such as a double-chain antibody fragment (dcFv or Fab) and a fully assembled antibody, in cells into which the library of expression vectors are introduced.
Type:
Grant
Filed:
October 31, 2000
Date of Patent:
August 26, 2003
Assignee:
Genetastix Corporation
Inventors:
Li Zhu, Shaobing Benjamin Hua, James Sheridan, Yu-Huei Lin
Abstract: A method for preparing saccharide compositions is disclosed. The method is reiterative and includes the following three steps.
(i) A glycosyltransferase capable of transferring a preselected saccharide unit to an acceptor moiety is isolated by contacting the acceptor moiety with a mixture suspected of containing the glycosyltransferase under conditions effective to bind the acceptor moiety and the glycosyltransferase and thereby isolate the glycosyltransferase. The acceptor moiety is a protein, a glycoprotein, a lipid, a glycolipid, or a carbohydrate.
(ii) The isolated glycosyltransferase is then used to catalyze the bond between the acceptor moiety and the preselected saccharide unit.
(iii) Steps (i) and (ii) are repeated a plurality of times with the intermediate product obtained in the first iteration of the method being used as the acceptor moiety of the second iteration.
Type:
Grant
Filed:
May 12, 2000
Date of Patent:
February 11, 2003
Assignee:
The Trustees of the University of Pennsylvania
Abstract: The present invention is directed to methods for producing gene targeting constructs by homologous recombination using mouse genomic libraries arrayed in yeast shuttle vectors. The invention is also directed to methods of using targeting constructs made by the methods to generate transgenic animals.
Abstract: Methods of shuffling nucleic acids to acquire or enhance mycotoxin detoxification activity, libraries of shuffled mycotoxin detoxification nucleic acids, transgenic cells, plants and DNA shuffling mixtures are provided.
Abstract: A random combinatorial selection method is disclosed for the construction of oligonucleotide aptamers in which nuclease resistance is conferred by the inclusion of modified nucleotides. The modified nucleotides are incorporated during PCR amplification to form achiral modified oligonucleotides. Thio-substituted aptamers are provided that bind tightly to the nuclear factor for human IL6 (NF-IL6).
Type:
Grant
Filed:
October 25, 1999
Date of Patent:
July 23, 2002
Assignee:
Board of Regents the University of Texas System
Inventors:
David G. Gorenstein, David J. King, Daniel A. Ventura, Allan R. Brasier