Altered and native immunoglobulins, including constant-variable region chimeras, are prepared in recombinant cell culture. The immunoglobulins contain variable regions which are immunologically capable of binding predetermined antigens. Methods are provided for refolding directly expressed immunoglobulins into immunologically active form.

Recombinant immunoglobin preparations

This invention provides methods of monitoring the expression levels of a multiplicity of genes. The methods involve hybridizing a nucleic acid sample to a high density array of oligonucleotide probes where the high density array contains oligonucleotide probes complementary to subsequences of target nucleic acids in the nucleic acid sample. In one embodiment, the method involves providing a pool of target nucleic acids comprising RNA transcripts of one or more target genes, or nucleic acids derived from the RNA transcripts, hybridizing said pool of nucleic acids to an array of oligonucleotide probes immobilized on surface, where the array comprising more than 100 different oligonucleotides and each different oligonucleotide is localized in a predetermined region of the surface, the density of the different oligonucleotides is greater than about 60 different oligonucleotides per 1 cm2, and the oligonucleotide probes are complementary to the RNA transcripts or nucleic acids derived from the RNA transcripts; and quantifying the hybridized nucleic acids in the array.

Expression monitoring by hybridization to high density oligonucleotide arrays

The invention pertains to a single polypeptide chain binding molecule which has binding specificity and affinity substantially similar to the binding specificity and affinity of the light and heavy chain aggregate variable region of an antibody, to genetic sequences coding therefor, and to recombinant DNA methods of producing such molecule and uses for such molecule.

Single polypeptide chain binding molecules

Inositol phospholipids and cell surface receptor function.

Polypeptide arrays can be synthesized on a substrate by attaching photoremovable groups to the surface of a substrate, exposing selected regions of the substrate to light to activate those regions, attaching an amino acid monomer with a photoremovable group to the activated regions, and repeating the steps of activation and attachment until polypeptides of the desired length and sequences are synthesized. The resulting array can be used to determine which peptides on the array can bind to a receptor.

Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element.

Preparation of bispecific antibodies by the chemical reassociation of monovalent fragments derived from monoclonal mouse immunoglobulin G1 is inefficient because of side reactions during reoxidation of the multiple disulfide bonds linking the heavy chains. These side reactions can be avoided by using specific dithiol complexing agents such as arsenite and effecting disulfide formation with a thiol activating agent such as 5,5'-dithiobis(2-nitrobenzoic acid). In this way bispecific antibodies were obtained in high yield and free of monospecific contaminants from monoclonal mouse immunoglobulin G1 fragments. The bispecific antibodies were used as agents for the selective immobilization of enzymes.

https://science.sciencemag.org/content/sci/229/4708/81.full.pdf

Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments

Protein splicing is a form of posttranslational processing that consists of the excision of an intervening polypeptide sequence, the intein, from a protein, accompanied by the concomitant joining of the flanking polypeptide sequences, the exteins, by a peptide bond. It requires neither cofactors nor auxiliary enzymes and involves a series of four intramolecular reactions, the first three of which occur at a single catalytic center of the intein. Protein splicing can be modulated by mutation and converted to highly specific self-cleavage and protein ligation reactions that are useful protein engineering tools. Some of the reactions characteristic of protein splicing also occur in other forms of protein autoprocessing, ranging from peptide bond cleavage to conjugation with nonprotein moieties. These mechanistic similarities may be the result of convergent evolution, but in at least one case-hedgehog protein autoprocessing-there is definitely a close evolutionary relationship to protein splicing.

Protein splicing and related forms of protein autoprocessing.

The enzymatic synthesis of inositol monophosphatide.

https://www.jbc.org/content/271/36/22159.full.pdf

Protein Splicing Involving the Saccharomyces cerevisiae VMA Intein THE STEPS IN THE SPLICING PATHWAY, SIDE REACTIONS LEADING TO PROTEIN CLEAVAGE, AND ESTABLISHMENT OF AN IN VITRO SPLICING SYSTEM

Henry Paulus

Papers

Single-column purification of free recombinant proteins using a self-cleavable affinity tag derived from a protein splicing element.

Preparation of bispecific antibodies by chemical recombination of monoclonal immunoglobulin G1 fragments

Protein splicing and related forms of protein autoprocessing.

The enzymatic synthesis of inositol monophosphatide.

Protein Splicing Involving the Saccharomyces cerevisiae VMA Intein THE STEPS IN THE SPLICING PATHWAY, SIDE REACTIONS LEADING TO PROTEIN CLEAVAGE, AND ESTABLISHMENT OF AN IN VITRO SPLICING SYSTEM